Polymorphic and amorphous forms of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide

ABSTRACT

Disclosed herein are polymorphic and amorphous forms of anhydrate, hydrate, and solvates of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide and methods of using such compositions for treating or suppressing oxidative stress disorders, including mitochondrial disorders, impaired energy processing disorders, neurodegenerative diseases and diseases of aging. Further disclosed are methods of making such polymorphic and amorphous forms.

The application is a continuation of U.S. patent application Ser. No.15/536,603, filed Jun. 15, 2017, entitled POLYMORPHIC AND AMORPHOUSFORMS OF(R)-2-HYDROXY-2-METHYL-4-(2,4,5-TRIMETHYL-3,6-DIOXOCYCLOHEXA-1,4-DIENYL)BUTANAMIDE,which is a National Stage filing under 35 U.S.C. 371 ofPCT/US2015/066211, filed Dec. 16, 2015, entitled POLYMORPHIC ANDAMORPHOUS FORMS OF(R)-2-HYDROXY-2-METHYL-4-(2,4,5-TRIMETHYL-3,6-DIOXOCYCLOHEXA-1,4-DIENYL)BUTANAMIDE,which claims priority to, and the benefit of, U.S. Provisional PatentApplication No. 62/092,743, filed Dec. 16, 2014, entitled POLYMORPHICAND AMORPHOUS FORMS OF(R)-2-HYDROXY-2-METHYL-4-(2,4,5-TRIMETHYL-3,6-DIOXOCYCLOHEXA-1,4-DIENYL)BUTANAMIDE,and U.S. Provisional Patent Application No. 62/133,276, filed Mar. 13,2015, entitled POLYMORPHIC AND AMORPHOUS FORMS OF(R)-2-HYDROXY-2-METHYL-4-(2,4,5-TRIMETHYL-3,6-DIOXOCYCLOHEXA-1,4-DIENYL)BUTANAMIDE,the contents of each of these applications are herein incorporated byreference in their entirety for all purposes.

TECHNICAL FIELD

The application discloses compositions and methods useful for treatmentor suppression of diseases, developmental delays and symptoms related tooxidative stress disorders. Examples of such disorders includemitochondrial disorders, impaired energy processing disorders,neurodegenerative diseases and diseases of aging. The applicationfurther discloses methods of making such compositions.

BACKGROUND

Oxidative stress is caused by disturbances to the normal redox statewithin cells. An imbalance between routine production and detoxificationof reactive oxygen species such as peroxides and free radicals canresult in oxidative damage to the cellular structure and machinery. Themost important source of reactive oxygen species under normal conditionsin aerobic organisms is probably the leakage of activated oxygen frommitochondria during normal oxidative respiration. Impairments associatedwith this process are suspected to contribute to mitochondrial disease,neurodegenerative disease, and diseases of aging.

Mitochondria are organelles in eukaryotic cells, popularly referred toas the “powerhouse” of the cell. One of their primary functions isoxidative phosphorylation. The molecule adenosine triphosphate (ATP)functions as an energy “currency” or energy carrier in the cell, andeukaryotic cells derive the majority of their ATP from biochemicalprocesses carried out by mitochondria. These biochemical processesinclude the citric acid cycle (the tricarboxylic acid cycle, or Krebscycle), which generates reduced nicotinamide adenine dinucleotide(NADH+H+) from oxidized nicotinamide adenine dinucleotide (NAD+), andoxidative phosphorylation, during which NADH+H+ is oxidized back toNAD+. The citric acid cycle also reduces flavin adenine dinucleotide, orFAD, to FADH2; FADH2 also participates in oxidative phosphorylation.

The electrons released by oxidation of NADH+H+ are shuttled down aseries of protein complexes (Complex I, Complex II, Complex III, andComplex IV) known as the mitochondrial respiratory chain. Thesecomplexes are embedded in the inner membrane of the mitochondrion.Complex IV, at the end of the chain, transfers the electrons to oxygen,which is reduced to water. The energy released as these electronstraverse the complexes is used to generate a proton gradient across theinner membrane of the mitochondrion, which creates an electrochemicalpotential across the inner membrane. Another protein complex, Complex V(which is not directly associated with Complexes I, II, III and IV) usesthe energy stored by the electrochemical gradient to convert ADP intoATP.

When cells in an organism are temporarily deprived of oxygen, anaerobicrespiration is utilized until oxygen again becomes available or the celldies. The pyruvate generated during glycolysis is converted to lactateduring anaerobic respiration. The buildup of lactic acid is believed tobe responsible for muscle fatigue during intense periods of activity,when oxygen cannot be supplied to the muscle cells. When oxygen againbecomes available, the lactate is converted back into pyruvate for usein oxidative phosphorylation.

Oxygen poisoning or toxicity is caused by high concentrations of oxygenthat may be damaging to the body and increase the formation offree-radicals and other structures such as nitric oxide, peroxynitrite,and trioxidane. Normally, the body has many defense systems against suchdamage but at higher concentrations of free oxygen, these systems areeventually overwhelmed with time, and the rate of damage to cellmembranes exceeds the capacity of systems which control or repair it.Cell damage and cell death then results.

Qualitative and/or quantitative disruptions in the transport of oxygento tissues result in energy disruption in the function of red cells andcontribute to various diseases such as haemoglobinopathies.Haemoglobinopathy is a kind of genetic defect that results in abnormalstructure of one of the globin chains of the hemoglobin molecule. Commonhaemoglobinopathies include thalassemia and sickle-cell disease.Thalassemia is an inherited autosomal recessive blood disease. Inthalassemia, the genetic defect results in reduced rate of synthesis ofone of the globin chains that make up hemoglobin. While thalassemia is aquantitative problem of too few globins synthesized, sickle-cell diseaseis a qualitative problem of synthesis of an incorrectly functioningglobin. Sickle-cell disease is a blood disorder characterized by redblood cells that assume an abnormal, rigid, and sickle shape. Sicklingdecreases the cells' flexibility and results in their restrictedmovement through blood vessels, depriving downstream tissues of oxygen.

Mitochondrial dysfunction contributes to various disease states. Somemitochondrial diseases are due to mutations or deletions in themitochondrial genome. If a threshold proportion of mitochondria in thecell is defective, and if a threshold proportion of such cells within atissue have defective mitochondria, symptoms of tissue or organdysfunction can result. Practically any tissue can be affected, and alarge variety of symptoms may be present, depending on the extent towhich different tissues are involved. Some examples of mitochondrialdiseases are Friedreich's ataxia (FRDA), Leber's Hereditary OpticNeuropathy (LHON), mitochondrial myopathy, encephalopathy, lactacidosis,and stroke (MELAS), Myoclonus Epilepsy Associated with Ragged-Red Fibers(MERRF) syndrome, Leigh's syndrome, and respiratory chain disorders.Most mitochondrial diseases involve children who manifest the signs andsymptoms of accelerated aging, including neurodegenerative diseases,stroke, blindness, hearing impairment, vision impairment, diabetes, andheart failure.

Friedreich's ataxia is an autosomal recessive neurodegenerative andcardiodegenerative disorder caused by decreased levels of the proteinFrataxin. The disease causes the progressive loss of voluntary motorcoordination (ataxia) and cardiac complications. Symptoms typicallybegin in childhood, and the disease progressively worsens as the patientgrows older; patients eventually become wheelchair-bound due to motordisabilities.

Leber's Hereditary Optic Neuropathy (LHON) is a disease characterized byblindness which occurs on average between 27 and 34 years of age. Othersymptoms may also occur, such as cardiac abnormalities and neurologicalcomplications.

Mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS)can manifest itself in infants, children, or young adults. Strokes,accompanied by vomiting and seizures, are one of the most serioussymptoms; it is postulated that the metabolic impairment of mitochondriain certain areas of the brain is responsible for cell death andneurological lesions, rather than the impairment of blood flow as occursin ischemic stroke.

Myoclonus Epilepsy Associated with Ragged-Red Fibers (MERRF) syndrome isone of a group of rare muscular disorders that are called mitochondrialencephalomyopathies. Mitochondrial encephalomyopathies are disorders inwhich a defect in the genetic material arises from a part of the cellstructure that releases energy (mitochondria). This can cause adysfunction of the brain and muscles (encephalomyopathies). Themitochondrial defect as well as “ragged-red fibers” (an abnormality oftissue when viewed under a microscope) are always present. The mostcharacteristic symptom of MERRF syndrome is myoclonic seizures that areusually sudden, brief, jerking, spasms that can affect the limbs or theentire body, difficulty speaking (dysarthria), optic atrophy, shortstature, hearing loss, dementia, and involuntary jerking of the eyes(nystagmus) may also occur.

Leigh's syndrome is a rare inherited neurometabolic disordercharacterized by degeneration of the central nervous system where thesymptoms usually begin between the ages of 3 months to 2 years andprogress rapidly. In most children, the first signs may be poor suckingability and loss of head control and motor skills. These symptoms may beaccompanied by loss of appetite, vomiting, irritability, continuouscrying, and seizures. As the disorder progresses, symptoms may alsoinclude generalized weakness, lack of muscle tone, and episodes oflactic acidosis, which can lead to impairment of respiratory and kidneyfunction. Heart problems may also occur.

Co-Enzyme Q10 Deficiency is a respiratory chain disorder, with syndromessuch as myopathy with exercise intolerance and recurrent myoglobin inthe urine manifested by ataxia, seizures or mental retardation andleading to renal failure (Di Mauro et al., (2005) Neuromusc. Disord.,15:311-315), “Childhood-onset cerebellar ataxia and cerebellar atrophy,”(Masumeci et al., (2001) Neurology 56:849-855 and Lamperti et al.,(2003) 60:1206:1208); and infantile encephalomyopathy associated withnephrosis. Biochemical measurement of muscle homogenates of patientswith CoQ10 deficiency showed severely decreased activities ofrespiratory chain complexes I and II+III, while complex IV (COX) wasmoderately decreased (Gempel et al., (2007) Brain, 130(8):2037-2044).

Complex I Deficiency or NADH dehydrogenase NADH-CoQ reductase deficiencyis a respiratory chain disorder, with symptoms classified by three majorforms: (1) fatal infantile multisystem disorder, characterized bydevelopmental delay, muscle weakness, heart disease, congenital lacticacidosis, and respiratory failure; (2) myopathy beginning in childhoodor in adult life, manifesting as exercise intolerance or weakness; and(3) mitochondrial encephalomyopathy (including MELAS), which may beginin childhood or adult life and consists of variable combinations ofsymptoms and signs, including ophthalmoplegia, seizures, dementia,ataxia, hearing loss, pigmentary retinopathy, sensory neuropathy, anduncontrollable movements.

Complex II Deficiency or Succinate dehydrogenase deficiency is arespiratory chain disorder with symptoms including encephalomyopathy andvarious manifestations, including failure to thrive, developmentaldelay, hypotonia, lethargy, respiratory failure, ataxia, myoclonus andlactic acidosis.

Complex III Deficiency or Ubiquinone-cytochrome C oxidoreductasedeficiency is a respiratory chain disorder with symptoms categorized infour major forms: (1) fatal infantile encephalomyopathy, congenitallactic acidosis, hypotonia, dystrophic posturing, seizures, and coma;(2) encephalomyopathies of later onset (childhood to adult life):various combinations of weakness, short stature, ataxia, dementia,hearing loss, sensory neuropathy, pigmentary retinopathy, and pyramidalsigns; (3) myopathy, with exercise intolerance evolving into fixedweakness; and (4) infantile histiocytoid cardiomyopathy.

Complex IV Deficiency or Cytochrome C oxidase deficiency is arespiratory chain disorder with symptoms categorized in two major forms:(1) encephalomyopathy, where patients typically are normal for the first6 to 12 months of life and then show developmental regression, ataxia,lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus, dystonia,pyramidal signs, respiratory problems and frequent seizures; and (2)myopathy with two main variants: (a) Fatal infantile myopathy—may beginsoon after birth and accompanied by hypotonia, weakness, lacticacidosis, ragged-red fibers, respiratory failure, and kidney problems:and (b) Benign infantile myopathy—may begin soon after birth andaccompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers,respiratory problems, but (if the child survives) followed byspontaneous improvement.

Complex V Deficiency or ATP synthase deficiency is a respiratory chaindisorder including symptoms such as slow, progressive myopathy.

CPEO or Chronic Progressive External Ophthalmoplegia Syndrome is arespiratory chain disorder including symptoms such as visual myopathy,retinitis pigmentosa, or dysfunction of the central nervous system.

Kearns-Sayre Syndrome (KSS) is a mitochondrial disease characterized bya triad of features including: (1) typical onset in persons younger thanage 20 years; (2) chronic, progressive, external ophthalmoplegia; and(3) pigmentary degeneration of the retina. In addition, KSS may includecardiac conduction defects, cerebellar ataxia, and raised cerebrospinalfluid (CSF) protein levels (e.g., >100 mg/dL). Additional featuresassociated with KSS may include myopathy, dystonia, endocrineabnormalities (e.g., diabetes, growth retardation or short stature, andhypoparathyroidism), bilateral sensorineural deafness, dementia,cataracts, and proximal renal tubular acidosis.

Maternally inherited diabetes and deafness (MIDD) is a mitochondrialdisorder characterized by maternally transmitted diabetes andsensorineural deafness. In most cases, MIDD is caused by a pointmutation in the mitochondrial gene MT-TL1, encoding the mitochondrialtRNA for leucine, and in rare cases in MT-TE and MT-TK genes, encodingthe mitochondrial tRNAs for glutamic acid, and lysine, respectively.

In addition to congenital disorders involving inherited defectivemitochondria, acquired mitochondrial dysfunction contributes todiseases, particularly neurodegenerative disorders associated with aginglike Parkinson's, Alzheimer's, and Huntington's Diseases. The incidenceof somatic mutations in mitochondrial DNA rises exponentially with age;diminished respiratory chain activity is found universally in agingpeople. Mitochondrial dysfunction is also implicated in excitoxic,neuronal injury, such as that associated with cerebrovascular accidents,seizures and ischemia.

Some of the above diseases appear to be caused by defects in Complex Iof the respiratory chain. Electron transfer from Complex I to theremainder of the respiratory chain is mediated by the compound coenzymeQ (also known as Ubiquinone). Oxidized coenzyme Q (CoQ_(ox) orUbiquinone) is reduced by Complex I to reduced coenzyme Q (CoQ_(red) orUbiquinol). The reduced coenzyme Q then transfers its electrons toComplex III of the respiratory chain, where it is re-oxidized toCoQ_(ox) (Ubiquinone). CoQ_(ox) can then participate in furtheriterations of electron transfer.

Very few treatments are available for patients suffering from thesemitochondrial diseases. Recently, the compound Idebenone has beenproposed for treatment of Friedreich's ataxia. While the clinicaleffects of Idebenone have been relatively modest, the complications ofmitochondrial diseases can be so severe that even marginally usefultherapies are preferable to the untreated course of the disease. Anothercompound, MitoQ, has been proposed for treating mitochondrial disorders(see U.S. Pat. No. 7,179,928); clinical results for MitoQ have not yetbeen reported. Administration of coenzyme Q10 (CoQ10) and vitaminsupplements has shown only transient beneficial effects in individualcases of KSS. CoQ10 supplementation has also been used for the treatmentof CoQ10 deficiency with mixed results.

Oxidative stress is suspected to be important in neurodegenerativediseases such as Motor Neuron Disease, Amyotrophic Lateral Sclerosis(ALS), Creutzfeldt-Jakob disease, Machado-Joseph disease,Spino-cerebellar ataxia, Multiple sclerosis (MS), Parkinson's disease,Alzheimer's disease, and Huntington's disease. Oxidative stress isthought to be linked to certain cardiovascular disease and also plays arole in the ischemic cascade due to oxygen reperfusion injury followinghypoxia. This cascade includes both strokes and heart attacks.

Damage accumulation theory, also known as the free radical theory ofaging, invokes random effects of free radicals produced during aerobicmetabolism that cause damage to DNA, lipids and proteins and accumulateover time. The concept of free radicals playing a role in the agingprocess was first introduced by Himan D (1956), “Aging—A theory based onfree-radical and radiation chemistry,” J. Gerontol. 11, 298-300.

According to the free radical theory of aging, the process of agingbegins with oxygen metabolism (Valko et al, (2004), “Role of oxygenradicals in DNA damage and cancer incidence,” Mol. Cell. Biochem., 266,37-56). Even under ideal conditions some electrons “leak” from theelectron transport chain. These leaking electrons interact with oxygento produce superoxide radicals, so that under physiological conditions,about 1-3% of the oxygen molecules in the mitochondria are convertedinto superoxide. The primary site of radical oxygen damage fromsuperoxide radical is mitochondrial DNA (mtDNA) (Cadenas et al., (2000)Mitochondrial free radical generation, oxidative stress and aging, FreeRadic. Res, 28, 601-609). The cell repairs much of the damage done tonuclear DNA (nDNA) but mtDNA repair seems to be less efficient.Therefore, extensive mtDNA damage accumulates over time and shuts downmitochondria causing cells to die and the organism to age.

Some of the diseases associated with increasing age are cancer, diabetesmellitus, hypertension, atherosclerosis, ischemia/reperfusion injury,rheumatoid arthritis, neurodegenerative disorders such as dementia,Alzheimer's and Parkinson's. Diseases resulting from the process ofaging as a physiological decline include decreases in muscle strength,cardiopulmonary function, vision and hearing as well as wrinkled skinand graying hair.

The ability to adjust biological production of energy has applicationsbeyond the diseases described above. Various other disorders can resultin suboptimal levels of energy biomarkers (sometimes also referred to asindicators of energetic function), such as ATP levels. Treatments forthese disorders are also needed, in order to modulate one or more energybiomarkers to improve the health of the patient. In other applications,it can be desirable to modulate certain energy biomarkers away fromtheir normal values in an individual that is not suffering from disease.For example, if an individual is undergoing an extremely strenuousundertaking, it can be desirable to raise the level of ATP in thatindividual.

Certain polymorphic or amorphous forms of a drug can have advantageouscharacteristics versus other forms; for example, increased stability,increased solubility, better handling properties, lack of associatedtoxic solvents, and increased purity.

Example 16 of PCT Application No. PCT/US2008/082374, published as WO2009/061744 on May 14, 2009, describes a synthesis for racemic2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide;this Example does not specifically describe the synthesis of anyparticular polymorphic or amorphous form for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideor any particular stereoisomer thereof.

BRIEF SUMMARY OF THE INVENTION

In one aspect of the invention is a polymorph of an anhydrate, ahydrate, or a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is selected from the group consisting of Form I,Form II, Form III, Form IV, Form V, and Form VI as described herein.

In another aspect of the invention is a polymorph of an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form I as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in FIG. 10. In some embodiments, a powder X-raydiffraction pattern for the polymorph comprises characteristic peaks atthe following angular positions, wherein the angular positions may varyby ±0.2: 12.06, 17.03, and 17.26. In some embodiments, a powder X-raydiffraction pattern for the polymorph comprises characteristic peaks atleast at the following angular positions, wherein the angular positionsmay vary by ±0.2:12.06, 17.03, and 17.26. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2: 12.06, 15.33, 17.03, and 17.26. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at least at the following angular positions,wherein the angular positions may vary by +0.2:12.06, 15.33, 17.03,17.26, and 18.72. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at thefollowing angular positions, where in the angular positions may vary by±0.2: 12.06, 15.33, 17.03, 17.26, and 18.72. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by ±0.2:7.67, 10.75, 12.06, 15.33, 16.41,17.03, 17.26, 18.72, 20.04, and 23.92. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:7.67, 10.75, 12.06, 15.33, 16.41, 17.03, 17.26, 18.72,20.04, 20.64, 20.91, 21.14, 22.58, 23.13, 23.92, 24.19, 24.53, 27.21,and 27.56. In some embodiments, a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at the following angularpositions, wherein the angular positions may vary by ±0.2:5.48, 7.67,10.75, 12.06, 15.33, 16.41, 17.03, 17.26, 17.71, 17.94, 18.40, 18.72,19.51, 20.04, 20.64, 20.91, 21.14, 21.55, 21.91, 22.25, 22.58, 23.13,23.41, 23.92, 24.19, 24.53, 25.64, 26.13, 26.34, 27.21, 27.56, 28.01,29.04, and 29.46. In some embodiments, including any of the foregoingembodiments, the angular positions may vary by ±0.1. In someembodiments, including any of the foregoing embodiments, the angularpositions may vary by ±0.05. In some embodiments, including any of theforegoing embodiments, the angular positions may vary by +0.02. In someembodiments, including any of the foregoing embodiments, the polymorphis isolated. In some embodiments, including any of the foregoingembodiments, the polymorph is present in a composition, wherein thecomposition is essentially free of Forms II-VI, wherein Forms II-VI aredescribed in Table A or Tables 3-7 respectively. In some embodiments,including any of the foregoing embodiments, the polymorph is present ina composition, wherein at least about 95% of the composition is thepolymorph, exclusive of any solvents, carriers or excipients.

In another aspect of the invention is a polymorph of an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form V as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in a) or b) of FIG. 30. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by +0.2:9.61, 11.49, and 15.45. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by +0.2:9.61, 11.49,and 15.45. In some embodiments, a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:9.61,11.49, 15.45, and 23.96. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at least at thefollowing angular positions, wherein the angular positions may vary by±0.2:9.61, 11.49, 14.80, 15.45, 23.96. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:9.61, 11.49, 12.93, 15.45, and 26.05. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:9.61, 11.49, 12.93,14.80, 15.45, 16.53, 23.96, 24.54, and 26.05. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by ±0.2:9.61, 11.49, 12.93, 14.80, 15.45,16.10, 16.34, 16.53, 20.18, 22.52, 22.86, 23.96, 24.54, and 26.05. Insome embodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:6.91, 7.72, 9.61, 11.49,11.86, 12.93, 13.19, 13.87, 14.80, 15.45, 16.10, 16.34, 16.53, 17.14,17.85, 19.12, 19.85, 20.18, 21.00, 22.06, 22.52, 22.86, 23.09, 23.96,24.54, 25.26, 26.05, and 26.90. In some embodiments, including any ofthe foregoing embodiments, the angular positions may vary by ±0.1. Insome embodiments, including any of the foregoing embodiments, theangular positions may vary by ±0.05. In some embodiments, including anyof the foregoing embodiments, the angular positions may vary by ±0.02.In some embodiments, including any of the foregoing embodiments, thepolymorph is isolated. In some embodiments, including any of theforegoing embodiments, the polymorph is present in a composition,wherein the composition is essentially free of Forms I-IV and -VI,wherein Forms I-IV and -VI are described in Table A or Tables 2, or 4-7respectively. In some embodiments, including any of the foregoingembodiments, the polymorph is present in a composition, wherein at leastabout 95% of the composition is the polymorph, exclusive of anysolvents, carriers or excipients.

In another aspect of the invention is a polymorph of a hydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form III as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in a) or b) of FIG. 20. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by ±0.2: 14.02, 15.23, and 21.10. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by ±0.2: 14.02, 15.23,and 21.10. In some embodiments, a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:9.16,14.02, 15.23, and 21.10. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at least at thefollowing angular positions, wherein the angular positions may vary by±0.2:9.16, 13.74, 14.02, 15.23, and 21.10. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:9.16, 14.02, 15.23, 21.10, and 22.69. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:9.16, 11.81, 13.74,14.02, 15.23, 21.10, 22.69, and 23.90. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:9.16, 11.81, 13.74, 14.02, 15.23, 17.35, 21.10, 22.69,23.15, 23.90, and 26.10. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at thefollowing angular positions, wherein the angular positions may vary by:0.2:9.16, 11.53, 11.81, 12.68, 12.93, 13.74, 14.02, 15.23, 16.53, 17.35,17.98, 18.54, 19.09, 20.23, 21.10, 21.93, 22.69, 23.15, 23.50, 23.90,24.65, 25.09, 25.46, 25.79, 26.10, 27.79, 28.22, 28.93, and 29.33. Insome embodiments, including any of the foregoing embodiments, theangular positions may vary by ±0.1. In some embodiments, including anyof the foregoing embodiments, the angular positions may vary by ±0.05.In some embodiments, including any of the foregoing embodiments, theangular positions may vary by ±0.02. In some embodiments, including anyof the foregoing embodiments, the polymorph is isolated. In someembodiments, including any of the foregoing embodiments, the polymorphis present in a composition, wherein the composition is essentially freeof Forms I, II, IV, V, and VI, wherein Forms I, II, IV, V, and VI aredescribed in Table A or Tables 2-3 and 5-7 respectively. In someembodiments, including any of the foregoing embodiments, the polymorphis present in a composition, wherein at least about 95% of thecomposition is the polymorph, exclusive of any solvents, carriers orexcipients.

In another aspect of the invention is a polymorph of a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form II as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in a) or b) of FIG. 15. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by ±0.2:9.63, 11.33, and 19.33. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by ±0.2:9.63, 11.33,19.33. In some embodiments, a powder X-ray diffraction pattern for thepolymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:9.63,11.33, 10.85, and 19.33. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at least at thefollowing angular positions, wherein the angular positions may vary by±0.2:9.63, 11.33, 10.85, 19.33, and 17.3. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:9.63, 10.85, 11.33, 13.47, and 19.33. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:5.76, 8.04, 9.63, 10.85,11.33, 11.97, 13.47, 14.75, 17.37, 17.71, and 19.33. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:5.76, 8.04, 9.63, 10.85,11.33, 11.97, 13.47, 14.75, 16.42, 16.89, 17.37, 17.71, 19.33, 22.89,and 24.59. In some embodiments, a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at the following angularpositions, wherein the angular positions may vary by ±0.2:5.76, 6.72,7.57, 8.04, 9.63, 10.85, 11.33, 11.97, 12.38, 13.13, 13.47, 14.75,15.28, 16.42, 16.89, 17.37, 17.71, 18.17, 18.66, 19.33, 20.01, 20.29,20.67, 20.90, 21.36, 21.54, 21.80, 22.55, 22.89, 23.27, 23.54, 23.87,24.35, 24.59, 24.87, 25.29, 25.55, 25.89, 26.44, 27.49, 28.01, 28.39,and 29.17. In some embodiments, including any of the foregoingembodiments, the angular positions may vary by ±0.1. In someembodiments, including any of the foregoing embodiments, the angularpositions may vary by ±0.05. In some embodiments, including any of theforegoing embodiments, the angular positions may vary by ±0.02. In someembodiments, including any of the foregoing embodiments, the polymorphis isolated. In some embodiments, including any of the foregoingembodiments, the polymorph is present in a composition, wherein thecomposition is essentially free of Forms I, and III-VI, wherein Forms I,and III—VI are described in Table A or Tables 2-4 and 6-7 respectively.In some embodiments, including any of the foregoing embodiments, thepolymorph is present in a composition, wherein at least about 95% of thecomposition is the polymorph, exclusive of any solvents, carriers orexcipients.

In another aspect of the invention is a polymorph of a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form IV as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in a), b), or c) of FIG. 25. In some embodiments,a powder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by +0.2:4.31, 12.97, and 13.20. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by ±0.2:4.31, 12.97,13.20. In some embodiments, a powder X-ray diffraction pattern for thepolymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:4.31,8.76, 12.97, and 13.20. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at least at thefollowing angular positions, wherein the angular positions may vary by±0.2:0.2:4.31, 8.76, 12.97, 13.20, 16.66. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by +0.2:4.31, 8.76, 12.97, 13.20, and 16.66. In someembodiments, a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at the following angular positions,wherein the angular positions may vary by ±0.2:4.31, 7.94, 8.76, 12.97,13.20, 16.66, 17.33, and 20.57. In some embodiments, a powder X-raydiffraction pattern for the polymorph comprises characteristic peaks atthe following angular positions, wherein the angular positions may varyby ±0.2:4.31, 7.94, 8.76, 12.97, 13.20, 15.08, 16.66, 17.33, 19.09,20.57, and 21.58. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at thefollowing angular positions, wherein the angular positions may vary by±0.2:4.31, 5.77, 6.28, 7.53, 7.94, 8.76, 9.39, 9.87, 10.54, 11.07,11.68, 12.02, 12.28, 12.97, 13.20, 13.52, 14.40, 15.08, 15.90, 16.66,16.96, 17.33, 17.59, 18.77, 19.09, 19.74, 20.27, 20.57, 21.09, 21.58,22.81, 23.23, 24.01, 24.65, and 25.60. In some embodiments, includingany of the foregoing embodiments, the angular positions may vary by±0.1. In some embodiments, including any of the foregoing embodiments,the angular positions may vary by ±0.05. In some embodiments, includingany of the foregoing embodiments, the angular positions may vary by±0.02. In some embodiments, including any of the foregoing embodiments,the polymorph is isolated. In some embodiments, including any of theforegoing embodiments, the polymorph is present in a composition,wherein the composition is essentially free of Forms I-III and V-VI,wherein Forms I-III and V—VI are described in Table A or Tables 2-5 and7 respectively. In some embodiments, including any of the foregoingembodiments, the polymorph is present in a composition, wherein at leastabout 95% of the composition is the polymorph, exclusive of anysolvents, carriers or excipients.

In another aspect of the invention is a polymorph of a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is Form VI as described herein. In someembodiments, the polymorph has a powder x-ray diffraction patternsubstantially as shown in a) of FIG. 33. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at the following angular positions, wherein the angular positionsmay vary by ±0.2:6.27, 9.91, and 12.94. In some embodiments, a powderX-ray diffraction pattern for the polymorph comprises characteristicpeaks at least at the following angular positions, wherein the angularpositions may vary by ±0.2:6.27, 9.91, and 12.94. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at least at the following angular positions,wherein the angular positions may vary by ±0.2:6.27, 9.91, 12.94, and15.71. In some embodiments, a powder X-ray diffraction pattern for thepolymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:6.27,9.91, 12.94, 15.71, and 19.13. In some embodiments, a powder X-raydiffraction pattern for the polymorph comprises characteristic peaks atthe following angular positions, wherein the angular positions may varyby ±0.2:6.27, 9.41, 9.91, 12.94, and 13.29. In some embodiments, apowder X-ray diffraction pattern for the polymorph comprisescharacteristic peaks at the following angular positions, wherein theangular positions may vary by ±0.2:6.27, 8.85, 9.41, 9.91, 12.94, 13.29,16.67, and 19.13. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at thefollowing angular positions, wherein the angular positions may vary by+0.2:4.39, 6.27, 8.85, 9.41, 9.91, 11.32, 12.94, 13.29, 14.03, 16.67,19.13, 20.76, and 22.06. In some embodiments, a powder X-ray diffractionpattern for the polymorph comprises characteristic peaks at thefollowing angular positions, wherein the angular positions may vary by+0.2:4.39, 6.27, 7.00, 8.62, 8.85, 9.41, 9.91, 11.32, 11.50, 12.25,12.56, 12.94, 13.29, 14.03, 14.82, 15.10, 15.44, 15.71, 16.01, 16.67,16.91, 17.33, 17.59, 18.33, 18.75, 19.13, 20.25, 20.76, 21.68, 22.06,22.27, 22.61, 22.94, 24.01, 24.33, 24.65, 25.48, 26.05, 28.63, and29.18. In some embodiments, including any of the foregoing embodiments,the angular positions may vary by ±0.1. In some embodiments, includingany of the foregoing embodiments, the angular positions may vary by±0.05. In some embodiments, including any of the foregoing embodiments,the angular positions may vary by ±0.02. In some embodiments, includingany of the foregoing embodiments, the polymorph is isolated. In someembodiments, including any of the foregoing embodiments, the polymorphis present in a composition, wherein the composition is essentially freeof Forms I-V, wherein Forms I-V are described in Table A or Tables 2-6respectively. In some embodiments, including any of the foregoingembodiments, the polymorph is present in a composition, wherein at leastabout 95% of the composition is the polymorph, exclusive of anysolvents, carriers or excipients.

In another aspect of the invention is a composition comprising amorphous(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.In some embodiments, the amorphous form is isolated. In someembodiments, the composition is essentially free of Forms 1-VI, whereinForms I-VI are described in Table A or Tables 2-7 respectively. In someembodiments, including any of the foregoing embodiments, at least about95% of the composition is amorphous(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,exclusive of any solvents, carriers or excipients.

In another aspect of the invention is a pharmaceutical compositioncomprising a polymorphic or amorphous form of an anhydrate, a hydrate,or a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,or composition comprising such form, as described herein, including anyof the foregoing or hereafter embodiments, and a pharmaceuticallyacceptable carrier. In some embodiments, the form is polymorph Form I.In some embodiments, the form is polymorph Form II. In some embodiments,the form is polymorph Form III. In some embodiments, the form ispolymorph Form IV. In some embodiments, the form is polymorph Form V. Insome embodiments, the form is polymorph Form VI. In some embodiments,the form is amorphous. In some embodiments, the pharmaceuticalcomposition has an HPLC purity of more than about 95% for the anhydrate,hydrate, or solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,exclusive of any solvents, carriers or excipients. In some embodiments,the pharmaceutical composition has an HPLC purity of more than about 99%for the anhydrate, hydrate, or solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,exclusive of any solvents, carriers or excipients. In some embodiments,the pharmaceutical composition has an HPLC purity of more than about99.9% for the anhydrate, hydrate, or solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,exclusive of any solvents, carriers or excipients. HPLC purity % refersto the proportional area of a given compound's HPLC peak with respect tothe area of all peaks in a given HPLC spectrum. HPLC % is calculated bydividing the area of a compound peak by the area of all peaks, in a HPLCspectrum, and multiplying this quotient by one-hundred.

In another aspect of the invention is a pharmaceutical compositioncomprising an active agent and a pharmaceutically acceptable carrier,wherein the active agent consists of, or consists essentially of, apolymorphic or amorphous form of an anhydrate, a hydrate, or a solvateof(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideas described herein. In some embodiments, the form is polymorph Form I.In some embodiments, the form is polymorph Form II. In some embodiments,the form is polymorph Form III. In some embodiments, the form ispolymorph Form IV. In some embodiments, the form is polymorph Form V. Insome embodiments, the form is polymorph Form VI. In some embodiments,the form is amorphous.

In another aspect of the invention is a method of treating orsuppressing an oxidative stress disorder, modulating one or more energybiomarkers, normalizing one or more energy biomarkers, or enhancing oneor more energy biomarkers, comprising administering to an individual inneed thereof a therapeutically effective amount or effective amount of apolymorphic or amorphous form of an anhydrate, a hydrate, or a solvateof(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,or composition comprising such form, as described herein, including anyof the foregoing or hereafter embodiments. The method can use anyindividual polymorphic or amorphous form of the invention as describedherein, or a combination of such. In some embodiments, the form ispolymorph Form I. In some embodiments, the form is polymorph Form II. Insome embodiments, the form is polymorph Form III. In some embodiments,the form is polymorph Form IV. In some embodiments, the form ispolymorph Form V. In some embodiments, the form is polymorph Form VI. Insome embodiments, the form is amorphous. In some embodiments, includingany of the foregoing embodiments, the anhydrate, hydrate, or solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideis administered in a pharmaceutical composition comprising thepolymorphic or amorphous form and a pharmaceutically acceptable carrier.In some embodiments, including any of the foregoing embodiments, thepharmaceutical composition comprises an active agent consistingessentially of the polymorphic or amorphous form of the anhydrate,hydrate, or solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.In some embodiments, including any of the foregoing embodiments, themethod is a method of treating or suppressing an oxidative stressdisorder. In some embodiments, including any of the foregoingembodiments, the method is a method of treating an oxidative stressdisorder. In some embodiments, including any of the foregoingembodiments, the method is a method of suppressing an oxidative stressdisorder. In some embodiments, including any of the foregoingembodiments, the oxidative stress disorder is selected from the groupconsisting of: a mitochondrial disorder; an inherited mitochondrialdisease; Alpers Disease; Barth syndrome; a Beta-oxidation Defect;Carnitine-Acyl-Carnitine Deficiency; Carnitine Deficiency; a CreatineDeficiency Syndrome; Co-Enzyme Q10 Deficiency; Complex I Deficiency;Complex II Deficiency; Complex III Deficiency; Complex IV Deficiency;Complex V Deficiency; COX Deficiency; chronic progressive externalophthalmoplegia (CPEO); CPT I Deficiency; CPT II Deficiency;Friedreich's Ataxia (FA); Glutaric Aciduria Type II; Kearns-SayreSyndrome (KSS); Lactic Acidosis; Long-Chain Acyl-CoA DehydrogenaseDeficiency (LCAD); LCHAD; Leigh's syndrome; Leigh-like Syndrome; Leber'sHereditary Optic Neuropathy (LHON); Lethal Infantile Cardiomyopathy(LIC); Luft Disease; Multiple Acyl-CoA Dehydrogenase Deficiency (MAD);Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCAD); MitochondrialMyopathy, Encephalopathy, Lactacidosis, Stroke (MELAS); MyoclonicEpilepsy with Ragged Red Fibers (MERRF); Mitochondrial Recessive AtaxiaSyndrome (MIRAS); Mitochondrial Cytopathy, Mitochondrial DNA Depletion;Mitochondrial Encephalopathy; Mitochondrial Myopathy;Myoneurogastrointestina Disorder and Encephalopathy (MNGIE); Neuropathy,Ataxia, and Retinitis Pigmentosa (NARP); Pearson Syndrome; PyruvateCarboxylase Deficiency; Pyruvate Dehydrogenase Deficiency; a POLGMutation; a Respiratory Chain Disorder; Short-Chain Acyl-CoADehydrogenase Deficiency (SCAD); SCHAD; Very Long-Chain Acyl-CoADehydrogenase Deficiency (VLCAD); a myopathy; cardiomyopathy;encephalomyopathy; a neurodegenerative disease; Parkinson's disease;Alzheimer's disease; amyotrophic lateral sclerosis (ALS); a motor neurondisease; a neurological disease; epilepsy; an age-associated disease;macular degeneration; diabetes; metabolic syndrome; cancer; braincancer; a genetic disease; Huntington's Disease; a mood disorder;schizophrenia; bipolar disorder; a pervasive developmental disorder;autistic disorder; Asperger's syndrome; childhood disintegrativedisorder (CDD); Rett's disorder; PDD—not otherwise specified (PDD-NOS);a cerebrovascular accident; stroke; a vision impairment; opticneuropathy; dominant inherited juvenile optic atrophy; optic neuropathycaused by a toxic agent; glaucoma; Stargardt's macular dystrophy;diabetic retinopathy; diabetic maculopathy; retinopathy of prematurity;ischemic reperfusion-related retinal injury; oxygen poisoning; ahaemoglobinopathy; thalassemia; sickle cell anemia; seizures; ischemia;renal tubular acidosis; attention deficit/hyperactivity disorder (ADHD);a neurodegenerative disorder resulting in hearing or balance impairment;Dominant Optic Atrophy (DOA); Maternally inherited diabetes and deafness(MIDD); chronic fatigue; contrast-induced kidney damage;contrast-induced retinopathy damage; Abetalipoproteinemia; retinitispigmentosum; Wolfram's disease; Tourette syndrome; cobalamin c defect;methylmalonic aciduria; glioblastoma; Down's syndrome; acute tubularnecrosis; a muscular dystrophy; a leukodystrophy; ProgressiveSupranuclear Palsy; spinal muscular atrophy; hearing loss; noise inducedhearing loss; traumatic brain injury; Juvenile Huntington's Disease;Multiple Sclerosis; NGLY1; Multiple System Atrophy;Adrenoleukodystrophy; and Adrenomyeloneuropathy. In some embodiments,the oxidative stress disorder is Multiple System Atrophy. In someembodiments, including any of the foregoing embodiments, the oxidativestress disorder is cancer. In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is bipolardisorder. In some embodiments, the oxidative stress disorder isschizophrenia. In some embodiments, including any of the foregoingembodiments, the oxidative stress disorder is an age-associated disease.In some embodiments, including any of the foregoing embodiments, theoxidative stress disorder is Huntington's Disease. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is Alzheimer's disease. In some embodiments, including any ofthe foregoing embodiments, the oxidative stress disorder is amyotrophiclateral sclerosis (ALS). In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is epilepsy. Insome embodiments, including any of the foregoing embodiments, theoxidative stress disorder is Parkinson's disease. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is seizures. In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is stroke. In someembodiments, including any of the foregoing embodiments, the oxidativestress disorder is a mitochondrial disorder. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is an inherited mitochondrial disease. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is Friedreich's Ataxia (FA). In some embodiments, including anyof the foregoing embodiments, the oxidative stress disorder isKearns-Sayre Syndrome (KSS). In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is Leigh Syndromeor Leigh-like Syndrome. In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is Leber'sHereditary Optic Neuropathy (LHON). In some embodiments, including anyof the foregoing embodiments, the oxidative stress disorder isMitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke (MELAS). Insome embodiments, including any of the foregoing embodiments, theoxidative stress disorder is Myoclonic Epilepsy with Ragged Red Fibers(MERRF). In some embodiments, including any of the foregoingembodiments, the oxidative stress disorder is macular degeneration. Insome embodiments, including any of the foregoing embodiments, theoxidative stress disorder is brain cancer. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is autistic disorder. In some embodiments, including any of theforegoing embodiments, the oxidative stress disorder is Rett's disorder.In some embodiments, including any of the foregoing embodiments, theoxidative stress disorder is Maternally inherited diabetes and deafness(MIDD). In some embodiments, including any of the foregoing embodiments,the oxidative stress disorder is chronic fatigue. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is contrast-induced kidney damage. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is contrast-induced retinopathy damage. In some embodiments,including any of the foregoing embodiments, the oxidative stressdisorder is cobalamin c defect. In some embodiments, including any ofthe foregoing embodiments, the method is a method for modulating one ormore energy biomarkers, normalizing one or more energy biomarkers, orenhancing one or more energy biomarkers, wherein the one or more energybiomarkers are selected from the group consisting of: lactic acid(lactate) levels, either in whole blood, plasma, cerebrospinal fluid, orcerebral ventricular fluid; pyruvic acid (pyruvate) levels, either inwhole blood, plasma, cerebrospinal fluid, or cerebral ventricular fluid;lactate/pyruvate ratios, either in whole blood, plasma, cerebrospinalfluid, or cerebral ventricular fluid; total, reduced or oxidizedglutathione levels, or reduced/oxidized glutathione ratio either inwhole blood, plasma, lymphocytes, cerebrospinal fluid, or cerebralventricular fluid; total, reduced or oxidized cysteine levels, orreduced/oxidized cysteine ratio either in whole blood, plasma,lymphocytes, cerebrospinal fluid, or cerebral ventricular fluid;phosphocreatine levels, NADH (NADH+H+) levels; NADPH (NADPH+H+) levels;NAD levels; NADP levels; ATP levels; reduced coenzyme Q (CoQ_(red))levels; oxidized coenzyme Q (CoQ_(ox)) levels; total coenzyme Q(CoQ_(tot)) levels; oxidized cytochrome C levels; reduced cytochrome Clevels; oxidized cytochrome C/reduced cytochrome C ratio; acetoacetatelevels, β hydroxy butyrate levels, acetoacetate/(3 hydroxy butyrateratio, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactiveoxygen species; levels of oxygen consumption (VO2); levels of carbondioxide output (VCO2); respiratory quotient (VCO2/VO2); exercisetolerance; and anaerobic threshold. Energy biomarkers can be measured inwhole blood, plasma, cerebrospinal fluid, cerebroventricular fluid,arterial blood, venous blood, or any other body fluid, body gas, orother biological sample useful for such measurement. In someembodiments, including any of the foregoing embodiments, the levels aremodulated to a value within about 2 standard deviations of the value ina healthy subject. In some embodiments, including any of the foregoingembodiments, the levels are modulated to a value within about 1 standarddeviation of the value in a healthy subject. In some embodiments,including any of the foregoing embodiments, the levels in a subject arechanged by at least about 10% above or below the level in the subjectprior to modulation. In some embodiments, including any of the foregoingembodiments, the levels are changed by at least about 20% above or belowthe level in the subject prior to modulation. In some embodiments,including any of the foregoing embodiments, the levels are changed by atleast about 30% above or below the level in the subject prior tomodulation. In some embodiments, including any of the foregoingembodiments, the levels are changed by at least about 40% above or belowthe level in the subject prior to modulation. In some embodiments,including any of the foregoing embodiments, the levels are changed by atleast about 50% above or below the level in the subject prior tomodulation. In some embodiments, including any of the foregoingembodiments, the levels are changed by at least about 75% above or belowthe level in the subject prior to modulation. In some embodiments,including any of the foregoing embodiments, the levels are changed by atleast about 100% above or at least about 90% below the level in thesubject prior to modulation. In some embodiments, including any of theforegoing embodiments, the subject or subjects in which a method oftreating or suppressing an oxidative stress disorder, modulating one ormore energy biomarkers, normalizing one or more energy biomarkers, orenhancing one or more energy biomarkers is performed is/are selectedfrom the group consisting of subjects undergoing strenuous or prolongedphysical activity; subjects with chronic energy problems; subjects withchronic respiratory problems; pregnant females; pregnant females inlabor; neonates; premature neonates; subjects exposed to extremeenvironments; subjects exposed to hot environments; subjects exposed tocold environments; subjects exposed to environments withlower-than-average oxygen content; subjects exposed to environments withhigher-than-average carbon dioxide content; subjects exposed toenvironments with higher-than-average levels of air pollution; airlinetravelers; flight attendants; subjects at elevated altitudes; subjectsliving in cities with lower-than-average air quality; subjects workingin enclosed environments where air quality is degraded; subjects withlung diseases; subjects with lower-than-average lung capacity;tubercular patients; lung cancer patients; emphysema patients; cysticfibrosis patients; subjects recovering from surgery; subjects recoveringfrom illness; elderly subjects; elderly subjects experiencing decreasedenergy; subjects suffering from chronic fatigue; subjects suffering fromchronic fatigue syndrome; subjects undergoing acute trauma; subjects inshock; subjects requiring acute oxygen administration; subjectsrequiring chronic oxygen administration; subjects requiring organvisualization via contrast solution; or other subjects with acute,chronic, or ongoing energy demands who can benefit from enhancement ofenergy biomarkers.

In another aspect of the invention is the use of a polymorphic oramorphous form of an anhydrate, a hydrate, or a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideas described herein, including any of the foregoing or hereafterdescribed embodiments, for treating or suppressing an oxidative stressdisorder. In some embodiments, the form is polymorph Form I. In someembodiments, the form is polymorph Form II. In some embodiments, theform is polymorph Form III. In some embodiments, the form is polymorphForm IV. In some embodiments, the form is polymorph Form V. In someembodiments, the form is polymorph Form VI. In some embodiments, theform is amorphous. In another aspect of the invention is the use of apolymorphic or amorphous form of an anhydrate, a hydrate, or a solvateof(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideas described herein, including any of the foregoing or hereafterdescribed embodiments, in the manufacture of a medicament for use intreating or suppressing an oxidative stress disorder. In someembodiments, the form is polymorph Form I. In some embodiments, the formis polymorph Form II. In some embodiments, the form is polymorph FormIII. In some embodiments, the form is polymorph Form IV. In someembodiments, the form is polymorph Form V. In some embodiments, the formis polymorph Form VI. In some embodiments, the form is amorphous.

For all compositions described herein, and all methods using acomposition described herein, the compositions can either comprise thelisted components or steps, or can “consist essentially of” the listedcomponents or steps. When a composition is described as “consistingessentially of” the listed components, the composition contains thecomponents listed, and may contain other components which do notsubstantially affect the condition being treated, but do not contain anyother components which substantially affect the condition being treatedother than those components expressly listed; or, if the compositiondoes contain extra components other than those listed whichsubstantially affect the condition being treated, the composition doesnot contain a sufficient concentration or amount of the extra componentsto substantially affect the condition being treated. When a method isdescribed as “consisting essentially of” the listed steps, the methodcontains the steps listed, and may contain other steps that do notsubstantially affect the condition being treated, but the method doesnot contain any other steps which substantially affect the conditionbeing treated other than those steps expressly listed. As a non-limitingspecific example, when a composition is described as ‘consistingessentially of’ a component, the composition may additionally containany amount of pharmaceutically acceptable carriers, vehicles, ordiluents and other such components which do not substantially affect thecondition being treated.

In another aspect of the invention is a process for the preparation ofpolymorph Form I of an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the process comprises the steps: (a) contacting(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidewith a liquid comprising IPA; and (b) removing the liquid. In someembodiments, step (a) comprises dissolving the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein the liquid. In some embodiments, step (a) comprises slurrying the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein the liquid. In some embodiments, the slurrying in step (a) may beperformed for at least about 24 hours. In some embodiments, includingany of the foregoing embodiments, the liquid is 100% IPA In someembodiments, including any of the foregoing embodiments, the liquid is98% IPA/2% water (v/v). In some embodiments, including any of theforegoing embodiments, the process further comprises step (a)(i): addingheptane to the liquid. In some embodiments, including any of theforegoing embodiments, step (b) comprises filtering the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.In some embodiments, including any of the foregoing embodiments, themixture in step (a) or step (a)(i) may be seeded with Form I crystals.In some embodiments, including any of the foregoing embodiments, the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein step (a) is at least about 95% pure. In various embodiments,including any of the foregoing embodiments, the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein step (a) is at least about 96%, at least about 97%, at least about98%, at least about 99%, at least about 99.5%, at least about 99.9%pure. In another aspect of the invention is an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideprepared by the above described process.

In another aspect of the invention is a process for the preparation ofpolymorph Form II of an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the process comprises the steps: (a) dissolving the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein EtOAc, (b) rapidly cooling the mixture from (a), and (c) isolatingthe(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.In some embodiments, the initial(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideis Form I. In some embodiments, including any of the foregoingembodiments, step (a) is at about 60° C. In some embodiments, includingany of the foregoing embodiments, step (b) comprises rapidly cooling themixture in an ice bath. In some embodiments, including any of theforegoing embodiments, the mixture in step (b) may be seeded with FormII crystals. In another aspect of the invention is an anhydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideprepared by the above described process.

In another aspect of the invention is a process for the preparation ofpolymorph Form III of a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the process comprises the steps: (a) combining(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand 0.5% MC/2% Tween 80 in water to create a slurry; (b) slurrying themixture from (a), and (c) removing the 0.5% MC/2% Tween 80 in water. Asused herein “MC” refers to methyl cellulose and “Tween 80” refers to acommercially available polysorbate nonionic surfactant In someembodiments, the initial(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideis Form I. In some embodiments, including any of the foregoingembodiments, step (b) is performed for at least about 24 hours. In someembodiments, including any of the foregoing embodiments, step (b) is atroom temperature. In some embodiments, including any of the foregoingembodiments, the mixture in step (b) may be seeded with Form IIIcrystals. In another aspect of the invention is a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideprepared by the above described process.

In another aspect of the invention is a process for the preparation ofpolymorph Form III of a solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the process comprises the steps: (a) combining(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand 0.5% MC in water to create a slurry; (b) slurrying the mixture from(a), and (c) removing the 0.5% MC in water. In some embodiments, theinitial(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideis Form I, II, IV, V or VI. In some embodiments, including any of theforegoing embodiments, step (b) is performed at room temperature. Insome embodiments, including any of the foregoing embodiments, step (b)may be performed for at least about 7 days. In some embodiments,including any of the foregoing embodiments, the mixture in step (b) maybe seeded with Form III crystals. In another aspect of the invention isa solvate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideprepared by the above described process.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideshort term slurry experiments, a) Pattern C, from slurry in 0.5% MethylCellulose/2% Tween 80, b) Pattern B, from slurry in tetrahydrofuran(THF), and c) starting material, Pattern A.

FIG. 2 shows a XRPD overlay of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidesample lots, a) starting material, Pattern A, and b) Pattern D followingevaporative crystallization from 2-methyltetrahydrofuran (2-MeTHF).

FIG. 3 shows a XRPD overlay of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidecrystallization experiments, a) starting material, Pattern A, b) PatternD following evaporative crystallization from 2-MeTHF (Example 4), c)from evaporative crystallization in 2-MeTHF (Example 5, fast cooling),and d) from evaporative crystallization in 2-MeTHF (Example 5, slowcooling).

FIG. 4 shows a XRPD stack plot of all(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideforms, a) starting material, Pattern A, b) Pattern B, c) Pattern C, d)Pattern D, e) Pattern E from fast cooling crystallization in ethylacetate (EtOAc), and f) Pattern F, from fast cooling crystallization in2-MeTHF.

FIG. 5 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideforms, a) Pattern E from fast cooling crystallization in EtOAc (Example5), and b) from scale-up fast cooling crystallization in EtOAc (Example6).

FIG. 6 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideforms, a) Pattern B from slurry in THF (Example 3), and b) from scale-upslurry in THF (Example 6).

FIG. 7 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideforms, a) Pattern C from slurry in 0.5% Methyl Cellulose/2% Tween 80(Example 3), and b) from scale-up slurry in 0.5% Methyl Cellulose/2%Tween 80 (Example 6).

FIG. 8 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidesamples, a) following 24 hours of competitive slurry in 0.5% MethylCellulose in water, b) starting material, Pattern A, c) Pattern C, andd) following 7 days of competitive slurry in 0.5% Methyl Cellulose inwater.

FIG. 9 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidesamples, a) following 24 hours of competitive slurry in IPA, b)following 7 days of competitive slurry in IPA, c) starting material,Pattern A, d) following 24 hours of competitive slurry in isopropanol(IPA)/2% water, e) following 7 days of competitive slurry in IPA/2%water.

FIG. 10 shows a XRPD of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A.

FIG. 11 shows an optical microscopy image of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A.

FIG. 12 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A.

FIG. 13 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A.

FIG. 14 shows a moisture sorption-desorption plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A.

FIG. 15 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern E isolated from single solvent fast cooling experiments, a) from50 mg scale and b) from 300 mg scale-up.

FIG. 16 shows an optical microscopy image of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern E.

FIG. 17 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern E.

FIG. 18 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern E.

FIG. 19 shows a moisture sorption-desorption plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern E.

FIG. 20 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern C from slurry in 0.5% Methyl Cellulose/2% Tween 80, a) from 50mg scale and b) from 300 mg scale-up.

FIG. 21 shows an optical microscopy image of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern C.

FIG. 22 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern C.

FIG. 23 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern C.

FIG. 24 shows a moisture sorption-desorption plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern C.

FIG. 25 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern B from slurry in THF, a) from 50 mg scale and b) from 300 mgscale-up.

FIG. 26 shows an optical microscopy image of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern B.

FIG. 27 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern B.

FIG. 28 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern B.

FIG. 29 shows a moisture sorption-desorption plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern B.

FIG. 30 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern D from evaporative crystallization in 2-MeTHF, a) from Example4, b) Example 5 (fast cooling), c) Example 5 (slow cooling) and d)starting material Pattern A.

FIG. 31 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern D.

FIG. 32 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern D.

FIG. 33 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern F from single solvent crystallization in 2-MeTHF, a) fromExample 6, b) following moisture sorption analysis (starting withPattern F from Example 6) and c) Pattern E.

FIG. 34 shows an optical microscopy image of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern F.

FIG. 35 shows a DSC thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern F.

FIG. 36 shows a TGA thermogram of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern F.

FIG. 37 shows a moisture sorption-desorption plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern F.

FIG. 38 shows (R)-2-hydroxy-2-methyl-4-(2,4,5-trim ethyl-3,6-di oxocyclohexa-1,4-dienyl)butanamide Polymorphic Form-Interrelations.

FIG. 39 shows a XRPD stack plot of Edison(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePolymorphic Forms.

FIG. 40 shows a ¹H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material, Pattern A

FIG. 41 shows a ¹H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern B.

FIG. 42 shows a ¹H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidePattern C

FIG. 43 shows a 1H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern D.

FIG. 44 shows a ¹H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern E

FIG. 45 shows a ¹H NMR spectrum of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,Pattern F.

DETAILED DESCRIPTION

The invention embraces polymorphic and amorphous forms of anhydrates,hydrates, and solvates of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideuseful in treating or suppressing diseases, developmental delays andsymptoms related to oxidative stress such as mitochondrial disorders,impaired energy processing disorders, neurodegenerative diseases anddiseases of aging, and methods of using such compositions for treatingor suppressing an oxidative stress disorder, or for modulating,normalizing, or enhancing one or more (e.g. one, two, three, or more)energy biomarkers. The invention further embraces methods for producingsuch polymorphic and amorphous forms.

The abbreviations used herein have their conventional meaning within thechemical and biological arts, unless otherwise specified.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The terms “a” or “an,” as used in herein means one or more, unlesscontext clearly dictates otherwise.

By “subject,” “individual,” or “patient” is meant an individualorganism, preferably a vertebrate, more preferably a mammal, mostpreferably a human.

(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidemay exist in anhydrate, hydrate, and solvate forms. Unless otherwisespecified or clear from context, as used herein the term“(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide”encompasses anhydrate, hydrate, and solvate forms of the compound.

The term “substantially as shown in” when referring, for example, to anXRPD pattern, a DSC thermogram, or a TGA graph includes a pattern,thermogram or graph that is not necessarily identical to those depictedherein, but that falls within the limits of experimental error ordeviations when considered by one of ordinary skill in the art. Forexample, in an XRPD pattern, the relative intensity of the peaks in thediffraction pattern can vary, e.g. due to sample preparation conditions.In addition, changes in temperature (when generating the XRPD data) canaffect the shape and location of peaks. The XRPD patterns given hereinwere generated at room temperature (˜25° C.). In some embodiments, theXRPD pattern is generated at about 15° C. to about 30° C. In someembodiments, the XRPD pattern is generated at about 20° C. to about 30°C. In some embodiments, the XRPD pattern is generated at about 23° C. toabout 27° C. In some embodiments, the XRPD pattern is generated at about24° C. to about 26° C. In some embodiments, the XRPD pattern isgenerated at about 25° C.

Similarly, when describing a polymorph by characteristic peaks (e.g.angular positions of the peaks), it is to be understood that thelocation of the peaks may vary depending on sample preparation,temperature, etc. The characteristic XRPD peaks given herein weregenerated at room temperature (˜25° C.). In some embodiments, the XRPDdata is generated at about 15° C. to about 30° C. In some embodiments,the XRPD data is generated at about 20° C. to about 30° C. In someembodiments, the XRPD data is generated at about 23° C. to about 27° C.In some embodiments, the XRPD data is generated at about 24° C. to about26° C. In some embodiments, the XRPD data is generated at about 25° C.

A polymorph or amorphous form that is “isolated” is used herein to referto a form that is at least 90% that particular form (i.e. less than 10%of the material is comprises of other forms or other compounds,including but not limited to(S)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

A polymorph composition that is “essentially free of” a particularcomponent(s) indicates that the composition contains less than about 5%of the particular component(s). As a non-limiting example, a polymorphcomposition that is essentially free of polymorph Form II indicates acomposition that contains less than about 5% of Form II. In someembodiments, “essentially free of” indicates that the compositioncontains less than about 4%, less than about 3%, less than about 2%, orless than about 1% of the particular component(s), or wherein theparticular component(s) are not present within the limit of detection.

“Angular positions” indicates Angle, 2 theta.

“Treating” a disorder with the compounds, compositions, and methodsdiscussed herein is defined as administering one or more of thecompounds or compositions discussed herein, with or without additionaltherapeutic agents, in order to reduce or eliminate either the disorderor one or more symptoms of the disorder, or to retard the progression ofthe disorder or of one or more symptoms of the disorder, or to reducethe severity of the disorder or of one or more symptoms of the disorder.“Suppression” of a disorder with the compounds, compositions, andmethods discussed herein is defined as administering one or more of thecompounds or compositions discussed herein, with or without additionaltherapeutic agents, in order to suppress the clinical manifestation ofthe disorder, or to suppress the manifestation of adverse symptoms ofthe disorder. The distinction between treatment and suppression is thattreatment occurs after adverse symptoms of the disorder are manifest ina subject, while suppression occurs before adverse symptoms of thedisorder are manifest in a subject. Suppression may be partial,substantially total, or total. Because some of the disorders areinherited, genetic screening can be used to identify patients at risk ofthe disorder. The compounds, compositions, and methods of the inventioncan then be administered to asymptomatic patients at risk of developingthe clinical symptoms of the disorder, in order to suppress theappearance of any adverse symptoms.

“Therapeutic use” of the compounds and compositions discussed herein isdefined as using one or more of the compounds or compositions discussedherein to treat or suppress a disorder, as defined above. An “effectiveamount” of a compound or composition is an amount of the compound orcomposition sufficient to modulate, normalize, or enhance one or moreenergy biomarkers (where modulation, normalization, and enhancement aredefined below). A “therapeutically effective amount” of a compound orcomposition is an amount of the compound or composition, which, whenadministered to a subject, is sufficient to reduce or eliminate either adisorder or one or more symptoms of a disorder, or to retard theprogression of a disorder or of one or more symptoms of a disorder, orto reduce the severity of a disorder or of one or more symptoms of adisorder, or to suppress the clinical manifestation of a disorder, or tosuppress the manifestation of adverse symptoms of a disorder. Atherapeutically effective amount can be given in one or moreadministrations. An “effective amount” of a compound or compositionembraces both a therapeutically effective amount, as well as an amounteffective to modulate, normalize, or enhance one or more energybiomarkers in a subject.

“Modulation” of, or to “modulate,” an energy biomarker means to changethe level of the energy biomarker towards a desired value, or to changethe level of the energy biomarker in a desired direction (e.g., increaseor decrease). Modulation can include, but is not limited to,normalization and enhancement as defined below.

“Normalization” of, or to “normalize,” an energy biomarker is defined aschanging the level of the energy biomarker from a pathological valuetowards a normal value, where the normal value of the energy biomarkercan be 1) the level of the energy biomarker in a healthy person orsubject, or 2) a level of the energy biomarker that alleviates one ormore undesirable symptoms in the person or subject. That is, tonormalize an energy biomarker which is depressed in a disease statemeans to increase the level of the energy biomarker towards the normal(healthy) value or towards a value which alleviates an undesirablesymptom; to normalize an energy biomarker which is elevated in a diseasestate means to decrease the level of the energy biomarker towards thenormal (healthy) value or towards a value which alleviates anundesirable symptom.

“Enhancement” of, or to “enhance,” energy biomarkers means tointentionally change the level of one or more energy biomarkers awayfrom either the normal value, or the value before enhancement, in orderto achieve a beneficial or desired effect. For example, in a situationwhere significant energy demands are placed on a subject, it may bedesirable to increase the level of ATP in that subject to a level abovethe normal level of ATP in that subject. Enhancement can also be ofbeneficial effect in a subject suffering from a disease or pathologysuch as e.g. a mitochondrial disorder, in that normalizing an energybiomarker may not achieve the optimum outcome for the subject; in suchcases, enhancement of one or more energy biomarkers can be beneficial,for example, higher-than-normal levels of ATP, or lower-than-normallevels of lactic acid (lactate) can be beneficial to such a subject.

By modulating, normalizing, or enhancing the energy biomarker Coenzyme Qis meant modulating, normalizing, or enhancing the variant or variantsof Coenzyme Q which is predominant in the species of interest. Forexample, the variant of Coenzyme Q which predominates in humans isCoenzyme Q10. If a species or subject has more than one variant ofCoenzyme Q present in significant amounts (i.e., present in amountswhich, when modulated, normalized, or enhanced, can have a beneficialeffect on the species or subject), modulating, normalizing, or enhancingCoenzyme Q can refer to modulating, normalizing or enhancing any or allvariants of Coenzyme Q present in the species or subject.

By “respiratory chain disorder” is meant a disorder which results in thedecreased utilization of oxygen by a mitochondrion, cell, tissue, orindividual, due to a defect or disorder in a protein or other componentcontained in the mitochondrial respiratory chain. By “protein or othercomponent contained in the mitochondrial respiratory chain” is meant thecomponents (including, but not limited to, proteins, tetrapyrroles, andcytochromes) comprising mitochondrial complex I, II, III, IV, and/or V.“Respiratory chain protein” refers to the protein components of thosecomplexes, and “respiratory chain protein disorder” is meant a disorderwhich results in the decreased utilization of oxygen by a mitochondrion,cell, tissue, or individual, due to a defect or disorder in a proteincontained in the mitochondrial respiratory chain.

The terms “Parkinson's”, (also called “Parkinsonism” and “Parkinsoniansyndrome”) (“PD”) is intended to include not only Parkinson's diseasebut also drug-induced Parkinsonism and post-encephalitic Parkinsonism.Parkinson's disease is also known as paralysis agitans or shaking palsy.It is characterized by tremor, muscular rigidity and loss of posturalreflexes. The disease usually progresses slowly with intervals of 10 to20 years elapsing before the symptoms cause incapacity. Due to theirmimicry of effects of Parkinson's disease, treatment of animals withmethamphetamine or MPTP has been used to generate models for Parkinson'sdisease. These animal models have been used to evaluate the efficacy ofvarious therapies for Parkinson's disease.

The term “Friedreich's ataxia” is intended to embrace other relatedataxias, and is also sometimes referred to as hereditary ataxia,familial ataxia, or Friedreich's tabes.

The term “ataxia” is an aspecific clinical manifestation implyingdysfunction of parts of the nervous system that coordinate movement,such as the cerebellum. People with ataxia have problems withcoordination because parts of the nervous system that control movementand balance are affected. Ataxia may affect the fingers, hands, arms,legs, body, speech, and eye movements. The word ataxia is often used todescribe a symptom of incoordination which can be associated withinfections, injuries, other diseases, or degenerative changes in thecentral nervous system. Ataxia is also used to denote a group ofspecific degenerative diseases of the nervous system called thehereditary and sporadic ataxias. Ataxias are also often associated withhearing impairments.

There are three types of ataxia, cerebellar ataxia, includingvestibulo-cerebellar dysfunction, spino-cerebellar dysfunction, andcerebro-cerebellar dysfunction; sensory ataxia; and vestibular ataxia.Examples of the diseases which are classifiable into spino-cerebellarataxia or multiple system atrophy are hereditary olivo-ponto-cerebellaratrophy, hereditary cerebellar cortical atrophy, Friedreich's ataxia,Machado-Joseph diseases, Ramsay Hunt syndrome, hereditarydentatorubral-pallidoluysian atrophy, hereditary spastic paraplegia,Shy-Drager syndrome, cortical cerebellar atrophy, striato-nigraldegeneration, Marinesco-Sjogren syndrome, alcoholic cortical cerebellaratrophy, paraneoplastic cerebellar atrophy associated with malignanttumor, toxic cerebellar atrophy caused by toxic substances, Vitamin Edeficiency due to mutation of a Tocopherol transfer protein (aTTP) orlipid absorption disorder such as Abetalipoproteinemia, cerebellaratrophy associated with endocrine disturbance and the like.

Examples of ataxia symptoms are motor ataxia, trunk ataxia, limb ataxiaand the like, autonomic disturbance such as orthostatic hypotension,dysuria, hypohidrosis, sleep apnea, orthostatic syncope and the like,stiffness of lower extremity, ocular nystagmus, oculomotor nervedisorder, pyramidal tract dysfunction, extrapyramidal symptoms (posturaladjustment dysfunction, muscular rigidity, akinesia, tremors),dysphagia, lingual atrophy, posterior funiculus symptom, muscle atrophy,muscle weakness, deep hyperreflexia, sensory disturbance, scoliosis,kyphoscoliosis, foot deformities, anarthria, dementia, manic state,decreased motivation for rehabilitation and the like.

Polymorphic and Amorphous Forms of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide

Provided herein are various crystalline and amorphous forms of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide:

and methods for producing such forms, and methods for using such forms.

Table 1 below provides a summary of certain polymorphic forms of theinvention of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

TABLE 1 Summary of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide Polymorphic Form Characterization ¹H NMR KF XRPD[Pattern] Form DSC TGA [Residual [Wt % Optical (Conditions) Designation[° C.] [% Wt. loss] Solvent] water] Microscopy Crystalline Form I 152.90.0 Consistent with 0.1 Birefringent [Pattern A] (Anhydrate) structure(Starting material) [0.28 wt % IPA] Crystalline Form IV 70.5, 4.7Consistent with 0.3 Birefringent [Pattern B] (THF 89.1, Structure (RTSlurry in THF) Solvate) 149.7 [6.9 wt % THF] Crystalline Form III 72.0,2.5, Consistent with 4.3 Birefringent [Pattern C] (Hydrate) 150.7 2.3structure (RT Slurry in 0.5% MC/2% Tween 80) Crystalline Form V 67.2,2.7, Consistent with — Birefringent [Pattern D] (2-MeTHF 92.2, 5.3Structure (Evap Cryst. In 2- Solvate) 150.6 [6.1 wt % 2- MeTHF) MeTHF]Crystalline Form II 133.9, 0.4 Consistent with 0.1 Birefringent [PatternE] (Anhydrate) 151.3 Structure [0.4 (Single Solvent fast wt. % EtOAc]cooling cryst. in EtOAc) Crystalline Form VI 93.2, 1.1, Consistent with0.1 Birefringent [Pattern F] (2-MeTHF 135.2 0.2 Structure [3.9 (Scaleup, Single Solvate) wt. % 2-MeTHF] Solvent fast cooling 151.0 cryst. in2-MeTHF)

FIG. 38 provides a chart showing interrelations between the variouspolymorphic forms for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.FIG. 39 shows a XRPD stack plot of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidepolymorphic forms.

Table A provides various embodiments of angular positions of certaincharacteristic peaks in powder x-ray diffraction for the polymorphicforms of the invention. In some embodiments, the angular positions mayvary by ±0.2. In some embodiments, the angular positions may vary by±0.1. In some embodiments, the angular positions may vary by ±0.05. Insome embodiments, the angular positions may vary by ±0.02.

TABLE A Angular Positions of Certain Characteristic Peaks in PowderX-ray Diffraction for Forms I-VI Angular Positions of CertainCharacteristic Form Peaks (Angle, 2 theta) (all ± 0.2) I Embodiment 1.12.06, 17.03, 17.26 Embodiment 2. 12.06, 15.33, 17.03, 17.26, 18.72Embodiment 3. 7.67, 10.75, 12.06, 15.33, 16.41, 17.03, 17.26, 18.72,20.04, 23.92 Embodiment 4. 7.67, 10.75, 12.06, 15.33, 16.41, 17.03,17.26, 18.72, 20.04, 20.64, 20.91, 21.14, 22.58, 23.13, 23.92, 24.19,24.53, 27.21, 27.56 Embodiment 5. 5.48, 7.67, 10.75, 12.06, 15.33,16.41, 17.03, 17.26, 17.71, 17.94, 18.40, 18.72, 19.51, 20.04, 20.64,20.91, 21.14, 21.55, 21.91, 22.25, 22.58, 23.13, 23.41, 23.92, 24.19,24.53, 25.64, 26.13, 26.34, 27.21, 27.56, 28.01, 29.04, 29.46 Embodiment6. 12.06, 15.33, 17.03, 17.26. Embodiment 7. 12.06, 15.33, 17.03, 17.26,18.72, 23.92. Embodiment 8. 12.06, 15.33, 17.03, 17.26, 18.72, 23.92,16.41 Embodiment 9. 12.06, 15.33, 17.03, 17.26, 18.72, 23.92, 16.41,10.75 Embodiment 10. 12.06, 15.33, 17.03, 17.26, 18.72, 23.92, 16.41,10.75, 20.64 V Embodiment 1. 9.61, 11.49, 15.45 Embodiment 2. 9.61,11.49, 12.93, 15.45, 23.96, 26.05 Embodiment 3. 9.61, 11.49, 12.93,14.80, 15.45, 16.53, 23.96, 24.54, 26.05 Embodiment 4. 9.61, 11.49,12.93, 14.80, 15.45, 16.10, 16.34, 16.53, 20.18, 22.52, 22.86, 23.96,24.54, 26.05 Embodiment 5. 6.91, 7.72, 9.61, 11.49, 11.86, 12.93, 13.19,13.87, 14.80, 15.45, 16.10, 16.34, 16.53, 17.14, 17.85, 19.12, 19.85,20.18, 21.00, 22.06, 22.52, 22.86, 23.09, 23.96, 24.54, 25.26, 26.05,26.90 Embodiment 6. 9.61, 11.49, 12.93, 15.45 Embodiment 7. 9.61, 11.49,12.93, 15.45, 23.96 Embodiment 8. 9.61, 11.49, 12.93, 15.45, 14.80Embodiment 9. 9.61, 11.49, 12.93, 15.45, 7.72 Embodiment 10. 9.61,11.49, 12.93, 15.45, 7.72, 16.53 III Embodiment 1. 14.02, 15.23, 21.10Embodiment 2. 9.16, 14.02, 15.23, 21.10, 22.69 Embodiment 3. 9.16,11.81, 13.74, 14.02, 15.23, 21.10, 22.69, 23.90 Embodiment 4. 9.16,11.81, 13.74, 14.02, 15.23, 17.35, 21.10, 22.69, 23.15, 23.90, 26.10Embodiments. 9.16, 11.53, 11.81, 12.68, 12.93, 13.74, 14.02, 15.23,16.53, 17.35, 17.98, 18.54, 19.09, 20.23, 21.10, 21.93, 22.69, 23.15,23.50, 23.90, 24.65, 25.09, 25.46, 25.79, 26.10, 27.79, 28.22, 28.93,29.33 Embodiment 6. 9.16, 14.02, 15.23, 21.10 Embodiment 7. 9.16, 13.74,14.02, 15.23, 21.10 Embodiment 8. 9.16, 11.81, 13.74, 14.02, 15.23,21.10 Embodiment 9. 9.16, 11.81, 13.74, 14.02, 15.23, 21.10, 23.90Embodiment 10. 9.16, 11.81, 13.74, 14.02, 15.23, 21.10, 22.69, 23.90 IIEmbodiment 1. 9.63, 11.33, 19.33 Embodiment 2. 9.63, 10.85, 11.33,13.47, 19.33 Embodiment 3. 5.76, 8.04, 9.63, 10.85, 11.33, 11.97, 13.47,14.75, 17.37, 17.71, 19.33 Embodiment 4. 5.76, 8.04, 9.63, 10.85, 11.33,11.97, 13.47, 14.75, 16.42, 16.89, 17.37, 17.71, 19.33, 22.89, 24.59Embodiment 5. 5.76, 6.72, 7.57, 8.04, 9.63, 10.85, 11.33, 11.97, 12.38,13.13, 13.47, 14.75, 15.28, 16.42, 16.89, 17.37, 17.71, 18.17, 18.66,19.33, 20.01, 20.29, 20.67, 20.90, 21.36, 21.54, 21.80, 22.55, 22.89,23.27, 23.54, 23.87, 24.35, 24.59, 24.87, 25.29, 25.55, 25.89, 26.44,27.49, 28.01, 28.39, 29.17 Embodiment 6. 9.63, 11.33, 10.85 Embodiment7. 9.63, 11.33, 10.85, 19.33 Embodiments. 9.63, 11.33, 10.85, 19.33,17.37 Embodiment 9. 9.63, 11.33, 10.85, 19.33, 17.37, 13.47 Embodiment10. 9.63, 11.33, 10.85, 19.33, 17.37, 13.47, 11.97 IV Embodiment 1.4.31, 12.97, 13.20 Embodiment 2. 4.31, 8.76, 12.97, 13.20, 16.66Embodiment 3. 4.31, 7.94, 8.76, 12.97, 13.20, 16.66, 17.33, 20.57Embodiment 4. 4.31, 7.94, 8.76, 12.97, 13.20, 15.08, 16.66, 17.33,19.09, 20.57, 21.58 Embodiment 5. 4.31, 5.77, 6.28, 7.53, 7.94, 8.76,9.39, 9.87, 10.54, 11.07, 11.68, 12.02, 12.28, 12.97, 13.20, 13.52,14.40, 15.08, 15.90, 16.66, 16.96, 17.33, 17.59, 18.77, 19.09, 19.74,20.27, 20.57, 21.09, 21.58, 22.81, 23.23, 24.01, 24.65, 25.60 Embodiment6. 12.97, 13.20, 8.76 Embodiment 7. 12.97, 13.20, 8.76, 16.66 Embodiment8. 12.97, 13.20, 8.76, 16.66, 4.31 Embodiment 9. 12.97, 13.20, 8.76,16.66, 4.31, 17.33 Embodiment 10. 12.97, 13.20, 8.76, 16.66, 4.31,17.33, 20.57 VI Embodiment 1. 6.27, 9.91, 12.94 Embodiment 2. 6.27,9.41, 9.91, 12.94, 13.29 Embodiment 3. 6.27, 8.85, 9.41, 9.91, 12.94,13.29, 16.67, 19.13 Embodiment 4. 4.39, 6.27, 8.85, 9.41, 9.91, 11.32,12.94, 13.29, 14.03, 16.67, 19.13, 20.76, 22.06 Embodiment 5. 4.39,6.27, 7.00, 8.62, 8.85, 9.41, 9.91, 11.32, 11.50, 12.25, 12.56, 12.94,13.29, 14.03, 14.82, 15.10, 15.44, 15.71, 16.01, 16.67, 16.91, 17.33,17.59, 18.33, 18.75, 19.13, 20.25, 20.76, 21.68, 22.06, 22.27, 22.61,22.94, 24.01, 24.33, 24.65, 25.48, 26.05, 28.63, 29.18 Embodiment 6.6.27, 9.91, 12.94, 15.71 Embodiment 7. 6.27, 9.91, 12.94 , 15.71, 19.13Embodiment 8. 6.27, 9.91, 12.94 , 15.71, , 16.91, 19.13 Embodiment 9.6.27, 9.41, 9.91, 12.94,15.71, , 16.91, 19.13 Embodiment 10. 6.27, 8.85,9.41, 9.91, 12.94, 15.71, , 16.91, 19.13

Tables 2-7 provide additional embodiments of angular positions ofcharacteristic peaks in powder x-ray diffraction for the polymorphicforms of the invention. In some embodiments, a polymorphic form ischaracterized by the angular positions of characteristics peaks shown inTable A. In some embodiments, the polymorphic form is characterized by 3or more (e.g. 4, 5, 6, 7, 8, 9, 10, or more than 10) angular positionsof characteristic peaks in powder x-ray diffraction as shown in Tables2-7 below. In some embodiments, the angular positions may vary by ±0.2.In some embodiments, the angular positions may vary by ±0.1. In someembodiments, the angular positions may vary by ±0.05. In someembodiments, the angular positions may vary by ±0.02.

As a non-limiting example, polymorph Form I may be characterized by 3,4, 5, 6, 7, 8, 9, 10, or more angular positions as shown in Table 2.

TABLE 2 Angular Positions of Characteristic Peaks in Powder X-rayDiffraction for Pattern A (Anhydrate Form I) Angle, 2 theta d spacing, A5.48 16.1 7.67 11.5 10.75 8.2 12.06 7.3 15.33 5.8 16.41 5.4 17.03 5.217.26 5.1 17.71 5.0 17.94 4.9 18.40 4.8 18.72 4.7 19.51 4.5 20.04 4.420.64 4.3 20.91 4.2 21.14 4.2 21.55 4.1 21.91 4.1 22.25 4.0 22.58 3.923.13 3.8 23.41 3.8 23.92 3.7 24.19 3.7 24.53 3.6 25.64 3.5 26.13 3.426.34 3.4 27.21 3.3 27.56 3.2 28.01 3.2 29.04 3.1 29.46 3.0

Polymorph Form I may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

In some examples, polymorph Form I is characterized by at least 3 ormore angular positions. In certain examples, these angular positionsinclude 12.06, 17.03, and 17.26±0.2. In some examples, polymorph Form Iis characterized by at least 4 or more angular positions. In certainexamples, these at least four angular positions include 12.1, 17.0,17.3, and 15.33±0.2. In certain examples, these at least four angularpositions include 12.1, 17.0, 17.3, 15.33, and 18.72±0.2.

TABLE 3 Angular Positions of Characteristic Peaks in Powder X- rayDiffraction for Pattern D (2-MeTHF Solvate Form V) Angle, 2 theta dspacing, A 6.91 12.8 7.72 11.4 9.61 9.2 11.49 7.7 11.86 7.5 12.93 6.813.19 6.7 13.87 6.4 14.80 6.0 15.45 5.7 16.10 5.5 16.34 5.4 16.53 5.417.14 5.2 17.85 5.0 19.12 4.6 19.85 4.5 20.18 4.4 21.00 4.2 22.06 4.022.52 3.9 22.86 3.9 23.09 3.8 23.96 3.7 24.54 3.6 25.26 3.5 26.05 3.426.90 3.3

Polymorph Form V may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

Polymorph Form III may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

TABLE 4 Angular Positions of Characteristic Peaks in Powder X-rayDiffraction for Pattern C (Hydrate Form III) Angle, 2 theta d spacing, A9.16 9.6 11.53 7.7 11.81 7.5 12.68 7.0 12.93 6.8 13.74 6.4 14.02 6.315.23 5.8 16.53 5.4 17.35 5.1 17.98 4.9 18.54 4.8 19.09 4.6 20.23 4.421.10 4.2 21.93 4.0 22.69 3.9 23.15 3.8 23.50 3.8 23.90 3.7 24.65 3.625.09 3.5 25.46 3.5 25.79 3.5 26.10 3.4 27.79 3.2 28.22 3.2 28.93 3.129.33 3.0

TABLE 5 Angular Positions of Characteristic Peaks in Powder X-rayDiffraction for Pattern E (Anhydrate II) Angle, 2 theta d spacing, A5.76 15.3 6.72 13.2 7.57 11.7 8.04 11.0 9.63 9.2 10.85 8.1 11.33 7.811.97 7.4 12.38 7.1 13.13 6.7 13.47 6.6 14.75 6.0 15.28 5.8 16.42 5.416.89 5.2 17.37 5.1 17.71 5.0 18.17 4.9 18.66 4.8 19.33 4.6 20.01 4.420.29 4.4 20.67 4.3 20.90 4.2 21.36 4.2 21.54 4.1 21.80 4.1 22.55 3.922.89 3.9 23.27 3.8 23.54 3.8 23.87 3.7 24.35 3.7 24.59 3.6 24.87 3.625.29 3.5 25.55 3.5 25.89 3.4 26.44 3.4 27.49 3.2 28.01 3.2 28.39 3.129.17 3.1

Polymorph Form II may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

TABLE 6 Angular Positions of Characteristic Peaks in Powder X-rayDiffraction for Pattern B (THF Solvate Form IV) Angle, 2 theta dspacing, A 4.31 20.5 5.77 15.3 6.28 14.1 7.53 11.7 7.94 11.1 8.76 10.19.39 9.4 9.87 9.0 10.54 8.4 11.07 8.0 11.68 7.6 12.02 7.4 12.28 7.212.97 6.8 13.20 6.7 13.52 6.5 14.40 6.1 15.08 5.9 15.90 5.6 16.66 5.316.96 5.2 17.33 5.1 17.59 5.0 18.77 4.7 19.09 4.6 19.74 4.5 20.27 4.420.57 4.3 21.09 4.2 21.58 4.1 22.81 3.9 23.23 3.8 24.01 3.7 24.65 3.625.60 3.5

Polymorph Form IV may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

TABLE 7 Angular Positions of Characteristic Peaks in Powder X- rayDiffraction for Pattern F (2-MeTHF Solvate Form VI) Angle, 2 theta dspacing, A 4.39 20.1 6.27 14.1 7.00 12.6 8.62 10.3 8.85 10.0 9.41 9.49.91 8.9 11.32 7.8 11.50 7.7 12.25 7.2 12.56 7.0 12.94 6.8 13.29 6.714.03 6.3 14.82 6.0 15.10 5.9 15.44 5.7 15.71 5.6 16.01 5.5 16.67 5.316.91 5.2 17.33 5.1 17.59 5.0 18.33 4.8 18.75 4.7 19.13 4.6 20.25 4.420.76 4.3 21.68 4.1 22.06 4.0 22.27 4.0 22.61 3.9 22.94 3.9 24.01 3.724.33 3.7 24.65 3.6 25.48 3.5 26.05 3.4 28.63 3.1 29.18 3.1

Polymorph Form VI may be characterized by 3, 4, 5, 6, 7, 8, 9, 10, ormore angular positions as shown below:

Certain polymorphic or amorphous forms of a drug can have advantageouscharacteristics versus other forms, which can affect the desirability ofthe drug from a pharmaceutical and/or manufacturing perspective, forexample: increased stability, increased solubility, better handlingproperties, lack of associated undesired solvents (e.g. solvates withtoxic solvents), increased purity, better particle size and/ordistribution, improved bulk density, and ease of manufacture.

Forms I, II, III and the amorphous form are anhydrates or hydrates, areadvantageously are not solvates with undesired solvents (e.g. THF and2-MeTHF).

Forms I-IV and VI were shown to have good solubilities in water(each >1.3 mg/ml), with Form I having the highest aqueous solubility at1.74 mg/ml (Example 10). Furthermore, Form I was shown to be soluble ina variety of polar and non-polar solvents (Example 2), indicating anability to be administered using a variety of solvents. In someembodiments, it is advantageous for a drug to have a physiological log Dclose to zero; solubility in polar and non-polar solvents thus indicatesa more favorable physiological log D. Form I was further shown to haveincreased solubility in a simple detergent (0.5% MC/2% Tween 80)(Example 2); such solubility in simple detergents may be advantageous,as these conditions may mimic gut conditions for oral administration ofthe drug. Form III (Hydrate), is formed under simple detergentconditions, and thus Form III may be the form of the drug that will beproduced in the gut.

Forms I, II, and III advantageously demonstrated stability to elevatedhumidity (Example 8). Form I was also tested by grinding, and showedstability to grinding (Example 9). As shown in the examples, theexperiments performed indicated that Form I was highly stable.

Forms that are non-hygroscopic are easier to handle from a manufacturingperspective; as shown in Example 11, Forms I, II, IV, and VI werenon-hygroscopic.

Certain particle shapes and sizes may be advantageous: particles thatare closer to spherical may be preferred, with plates and needles lesspreferred. As shown in Example 11, Forms I, II, and III had morefavorable shapes, whereas Form VI was plate shaped, and Form IVneedle-shaped. Regarding particle size, smaller, more homogenous sizesmay be preferred. Smaller particles may have increased bioavailability,may be easier to dissolve, and may be easier to handle due to decreaseddrying times. Furthermore, smaller particles may not require amicronizing step that may be required for larger particles. As shown inthe Figures, Forms I-III had more favorable particle sizes than IV andVI.

Higher melting points may indicate a form with improved handlingcharacteristics (e.g. easier to dry and process) and more thermalstability. Form I had the highest melting point of Forms I-VI.Furthermore, a single peak may in some embodiments be preferred, asmultiple peaks may indicate conversion to a different form. Form I had asingle DSC peak, all others had two or three.

The various forms (polymorphs and amorphous) of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidemay also be utilized as intermediates in making a desired form. As anon-limiting example, if a preferred synthetic method for making(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideresults in a non-preferred form, the non-preferred form may be utilizedas an intermediate to make the desired form.

Diseases Amenable to Treatment or Suppression with Compositions andMethods of the Invention

A variety of disorders/diseases are believed to be caused or aggravatedby oxidative stress affecting normal electron flow in the cells, such asmitochondrial disorders, impaired energy processing disorders,neurodegenerative diseases and diseases of aging, and can be treated orsuppressed using the polymorphic and amorphous forms of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand methods of the invention.

Non-limiting examples of oxidative stress disorders include, forexample, mitochondrial disorders (including inherited mitochondrialdiseases) such as Alpers Disease, Barth syndrome, Beta-oxidationDefects, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency,Creatine Deficiency Syndromes, Co-Enzyme Q10 Deficiency, Complex IDeficiency, Complex II Deficiency, Complex III Deficiency, Complex IVDeficiency, Complex V Deficiency, COX Deficiency, chronic progressiveexternal ophthalmoplegia (CPEO), CPT I Deficiency, CPT II Deficiency,Friedreich's Ataxia (FA), Glutaric Aciduria Type II, Kearns-SayreSyndrome (KSS), Lactic Acidosis, Long-Chain Acyl-CoA DehydrogenaseDeficiency (LCAD), LCHAD, Leigh Disease or Syndrome, Leigh-likeSyndrome, Leber's Hereditary Optic Neuropathy (LHON, also referred to asLeber's Disease, Leber's Optic Atrophy (LOA), or Leber's OpticNeuropathy (LON)), Lethal Infantile Cardiomyopathy (LIC), Luft Disease,Multiple Acyl-CoA Dehydrogenase Deficiency (MAD), Medium-Chain Acyl-CoADehydrogenase Deficiency (MCAD), Mitochondrial Myopathy, Encephalopathy,Lactacidosis, Stroke (MELAS), Myoclonic Epilepsy with Ragged Red Fibers(MERRF), Mitochondrial Recessive Ataxia Syndrome (MIRAS), MitochondrialCytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathy,Mitochondrial Myopathy, Myoneurogastrointestinal Disorder andEncephalopathy (MNGIE), Neuropathy, Ataxia, and Retinitis Pigmentosa(NARP), Pearson Syndrome, Pyruvate Carboxylase Deficiency, PyruvateDehydrogenase Deficiency, POLG Mutations, Respiratory Chain Disorder,Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCAD), SCHAD, VeryLong-Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD); myopathies such ascardiomyopathy and encephalomyopathy; neurodegenerative diseases such asParkinson's disease, Alzheimer's disease, and amyotrophic lateralsclerosis (ALS, also known as Lou Gehrig's disease); motor neurondiseases; neurological diseases such as epilepsy; age-associateddiseases, particularly diseases for which CoQ10 has been proposed fortreatment, such as macular degeneration, diabetes (e.g. Type 2 diabetesmellitus), metabolic syndrome, and cancer (e.g. brain cancer); geneticdiseases such as Huntington's Disease (which is also a neurologicaldisease); mood disorders such as schizophrenia and bipolar disorder;pervasive developmental disorders such as autistic disorder, Asperger'ssyndrome, childhood disintegrative disorder (CDD), Rett's disorder, andPDD—not otherwise specified (PDD-NOS); cerebrovascular accidents such asstroke; vision impairments such as those caused by neurodegenerativediseases of the eye such as optic neuropathy, Leber's hereditary opticneuropathy, dominant inherited juvenile optic atrophy, optic neuropathycaused by toxic agents, glaucoma, age-related macular degeneration (both“dry” or non-exudative macular degeneration and “wet” or exudativemacular degeneration), Stargardt's macular dystrophy, diabeticretinopathy, diabetic maculopathy, retinopathy of prematurity, orischemic reperfusion-related retinal injury; disorders caused by energyimpairment include diseases due to deprivation, poisoning or toxicity ofoxygen, and qualitative or quantitative disruption in the transport ofoxygen such as haemoglobionopathies, for example thalassemia or sicklecell anemia; other diseases in which mitochondrial dysfunction isimplicated such as excitoxic, neuronal injury, such as that associatedwith seizures, stroke and ischemia; and other disorders including renaltubular acidosis; attention deficit/hyperactivity disorder (ADHD);neurodegenerative disorders resulting in hearing or balance impairment;Dominant Optic Atrophy (DOA); Maternally inherited diabetes and deafness(MIDD); chronic fatigue; contrast-induced kidney damage;contrast-induced retinopathy damage; Abetalipoproteinemia; retinitispigmentosum; Wolfram's disease; Tourette syndrome; cobalamin c defect;methylmalonic aciduria; glioblastoma; Down's syndrome; acute tubularnecrosis; muscular dystrophies; leukodystrophies; ProgressiveSupranuclear Palsy; spinal muscular atrophy; hearing loss (e.g. noiseinduced hearing loss); traumatic brain injury; Juvenile Huntington'sDisease; Multiple Sclerosis; NGLY1; Multiple System Atrophy;Adrenoleukodystrophy; and Adrenomyeloneuropathy. It is to be understoodthat certain specific diseases or disorders may fall within more thanone category; for example, Huntington's Disease is a genetic disease aswell as a neurological disease. Furthermore, certain oxidative stressdiseases and disorders may also be considered mitochondrial disorders.

For some disorders amenable to treatment with compounds and methods ofthe invention, the primary cause of the disorder is due to a defect inthe respiratory chain or another defect preventing normal utilization ofenergy in mitochondria, cells, or tissue(s). Non-limiting examples ofdisorders falling in this category include inherited mitochondrialdiseases, such as Myoclonic Epilepsy with Ragged Red Fibers (MERRF),Mitochondrial Myopathy, Encephalopathy, Lactacidosis, and Stroke(MELAS), Leber's Hereditary Optic Neuropathy (LHON, also referred to asLeber's Disease, Leber's Optic Atrophy (LOA), or Leber's OpticNeuropathy (LON)), Leigh Disease or Leigh Syndrome, Kearns-SayreSyndrome (KSS), and Friedreich's Ataxia (FA). For some disordersamenable to treatment with compounds and methods of the invention, theprimary cause of the disorder is not due to respiratory chain defects orother defects preventing normal utilization of energy in mitochondria,cells, or tissue(s); non-limiting examples of disorders falling in thiscategory include stroke, cancer, and diabetes. However, these latterdisorders are particularly aggravated by energy impairments, and areparticularly amenable to treatment with compounds of the invention inorder to ameliorate the condition. Pertinent examples of such disordersinclude ischemic stroke and hemorrhagic stroke, where the primary causeof the disorder is due to impaired blood supply to the brain. While anischemic episode caused by a thrombosis or embolism, or a hemorrhagicepisode caused by a ruptured blood vessel, is not primarily caused by adefect in the respiratory chain or another metabolic defect preventingnormal utilization of energy, oxidative stress plays a role in theischemic cascade due to oxygen reperfusion injury following hypoxia(this cascade occurs in heart attacks as well as in strokes).Accordingly, treatment with compounds and methods of the invention willmitigate the effects of the disease, disorder or condition. Modulatingone or more energy biomarkers, normalizing one or more energybiomarkers, or enhancing one or more energy biomarkers can also provebeneficial in such disorders both as a therapeutic measure and aprophylactic measure. For example, for a patient scheduled to undergonon-emergency repair of an aneurysm, enhancing energy biomarkers beforeand during the pre-operative can improve the patient's prognosis shouldthe aneurysm rupture before successful repair.

The term “oxidative stress disorder” or “oxidative stress disease”encompass both diseases caused by oxidative stress and diseasesaggravated by oxidative stress. The terms “oxidative stress disorder” or“oxidative stress disease” encompass both diseases and disorders wherethe primary cause of the disease is due to a defect in the respiratorychain or another defect preventing normal utilization of energy inmitochondria, cells, or tissue(s), and also diseases and disorders wherethe primary cause of the disease is not due to a defect in therespiratory chain or another defect preventing normal utilization ofenergy in mitochondria, cells, or tissue(s). The former set of diseasescan be referred to as “primary oxidative stress disorders,” while thelatter can be referred to as “secondary oxidative stress disorders.” Itshould be noted that the distinction between “diseases caused byoxidative stress” and “diseases aggravated by oxidative stress” is notabsolute; a disease may be both a disease caused by oxidative stress anda disease aggravated by oxidative stress. The boundary between “primaryoxidative stress disorder” and a “secondary oxidative stress disorder”is more distinct, provided that there is only one primary cause of adisease or disorder and that primary cause is known.

Bearing in mind the somewhat fluid boundary between diseases caused byoxidative stress and diseases aggravated by oxidative stress,mitochondrial diseases or disorders and impaired energy processingdiseases and disorders tend to fall into the category of diseases causedby oxidative stress, while neurodegenerative disorders and diseases ofaging tend to fall into the category of diseases aggravated by oxidativestress. Mitochondrial diseases or disorders and impaired energyprocessing diseases and disorders are generally primary oxidative stressdisorders, while neurodegenerative disorders and diseases of aging maybe primary or secondary oxidative stress disorders

Clinical Assessment of Oxidative Stress and Efficacy of Therapy

Several readily measurable clinical markers are used to assess themetabolic state of patients with oxidative stress disorders. Thesemarkers can also be used as indicators of the efficacy of a giventherapy, as the level of a marker is moved from the pathological valueto the healthy value. These clinical markers include, but are notlimited to, energy biomarkers such as lactic acid (lactate) levels,either in whole blood, plasma, cerebrospinal fluid, or cerebralventricular fluid; pyruvic acid (pyruvate) levels, either in wholeblood, plasma, cerebrospinal fluid, or cerebral ventricular fluid;lactate/pyruvate ratios, either in whole blood, plasma, cerebrospinalfluid, or cerebral ventricular fluid; total, reduced or oxidizedglutathione levels, or reduced/oxidized glutathione ratio either inwhole blood, plasma, lymphocytes, cerebrospinal fluid, or cerebralventricular fluid; total, reduced or oxidized cysteine levels, orreduced/oxidized cysteine ratio either in whole blood, plasma,lymphocytes, cerebrospinal fluid, or cerebral ventricular fluid;phosphocreatine levels, NADH (NADH+H+) or NADPH (NADPH+H+) levels; NADor NADP levels; ATP levels; anaerobic threshold; reduced coenzyme Q(CoQ_(red)) levels; oxidized coenzyme Q (CoQ_(ox)) levels; totalcoenzyme Q (CoQ_(tot)) levels; oxidized cytochrome C levels; reducedcytochrome C levels; oxidized cytochrome C/reduced cytochrome C ratio;acetoacetate levels, β-hydroxy butyrate levels, acetoacetate/β-hydroxybutyrate ratio, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels ofreactive oxygen species; and levels of oxygen consumption (VO2), levelsof carbon dioxide output (VCO2), and respiratory quotient (VCO2/VO2).Several of these clinical markers are measured routinely in exercisephysiology laboratories, and provide convenient assessments of themetabolic state of a subject. In one embodiment of the invention, thelevel of one or more energy biomarkers in a patient suffering from anoxidative stress disorder, such as Friedreich's ataxia, Leber'shereditary optic neuropathy, MELAS, KSS or CoQ10 deficiency, is improvedto within two standard deviations of the average level in a healthysubject. In another embodiment of the invention, the level of one ormore of these energy biomarkers in a patient suffering from an oxidativestress disorder, such as Friedreich's ataxia, Leber's hereditary opticneuropathy, MELAS, KSS or CoQ10 deficiency is improved to within onestandard deviation of the average level in a healthy subject. Exerciseintolerance can also be used as an indicator of the efficacy of a giventherapy, where an improvement in exercise tolerance (i.e., a decrease inexercise intolerance) indicates efficacy of a given therapy.

Several metabolic biomarkers have already been used to evaluate efficacyof CoQ10, and these metabolic biomarkers can be monitored as energybiomarkers for use in the methods of the current invention. Lactate, aproduct of the anaerobic metabolism of glucose, is removed by reductionto pyruvate in an aerobic setting or by oxidative metabolism, which isdependent on a functional mitochondrial respiratory chain. Dysfunctionof the respiratory chain may lead to inadequate removal of lactate andpyruvate from the circulation and elevated lactate/pymvate ratios areobserved in mitochondrial cytopathies (see Scriver C R, The metabolicand molecular bases of inherited disease, 7th ed., New York:McGraw-Hill, Health Professions Division, 1995; and Munnich et al., J.Inherit. Metab. Dis. 15(4):448-55 (1992)). Blood lactate/pyruvate ratio(Chariot et al., Arch. Pathol. Lab. Med. 118(7):695-7 (1994)) is,therefore, widely used as a noninvasive test for detection ofmitochondrial cytopathies (see again Scriver C R, The metabolic andmolecular bases of inherited disease, 7th ed., New York: McGraw-Hill,Health Professions Division, 1995; and Munnich et al., J. Inherit.Metab. Dis. 15(4):448-55 (1992)) and toxic mitochondrial myopathies(Chariot et al., Arthritis Rheum. 37(4):583-6 (1994)). Changes in theredox state of liver mitochondria can be investigated by measuring thearterial ketone body ratio (acetoacetate/3-hydroxybutyrate:AKBR) (Uedaet al., J. Cardiol. 29(2):95-102 (1997)). Urinary excretion of8-hydroxy-2′-deoxyguanosine (8-OHdG) often has been used as a biomarkerto assess the extent of repair of ROS-induced DNA damage in bothclinical and occupational settings (Erhola et al., FEBS Lett.409(2):287-91 (1997); Honda et al., Leuk. Res. 24(6):461-8 (2000);Pilger et al., Free Radic. Res. 35(3):273-80 (2001); Kim et al. EnvironHealth Perspect 112(6):666-71 (2004)).

Magnetic resonance spectroscopy (MRS) has been useful in the diagnosesof mitochondrial cytopathy by demonstrating elevations in cerebrospinalfluid (CSF) and cortical white matter lactate using proton MRS (1H-MRS)(Kaufmann et al., Neurology 62(8): 1297-302 (2004)). Phosphorous MRS(31P-MRS) has been used to demonstrate low levels of corticalphosphocreatine (PCr) (Matthews et al., Ann. Neurol. 29(4):435-8(1991)), and a delay in PCr recovery kinetics following exercise inskeletal muscle (Matthews et al., Ann. Neurol. 29(4):435-8 (1991);Barbiroli et al., J. Neurol. 242(7):472-7 (1995); Fabrizi et al., J.Neurol. Sci. 137(1):20-7 (1996)). A low skeletal muscle PCr has alsobeen confirmed in patients with mitochondrial cytopathy by directbiochemical measurements.

Exercise testing is particularly helpful as an evaluation and screeningtool in mitochondrial myopathies. One of the hallmark characteristics ofmitochondrial myopathies is a reduction in maximal whole body oxygenconsumption (VO2max) (Taivassalo et al., Brain 126(Pt 2):413-23 (2003)).Given that VO2max is determined by cardiac output (Qc) and peripheraloxygen extraction (arterial-venous total oxygen content) difference,some mitochondrial cytopathies affect cardiac function where deliverycan be altered; however, most mitochondrial myopathies show acharacteristic deficit in peripheral oxygen extraction (A-V O2difference) and an enhanced oxygen delivery (hyperkinetic circulation)(Taivassalo et al., Brain 126(Pt 2):413-23 (2003)). This can bedemonstrated by a lack of exercise induced deoxygenation of venous bloodwith direct AV balance measurements (Taivassalo et al., Ann. Neurol.51(1):38-44 (2002)) and non-invasively by near infrared spectroscopy(Lynch et al., Muscle Nerve 25(5):664-73 (2002); van Beekvelt et al.,Ann. Neurol. 46(4):667-70 (1999)).

Several of these energy biomarkers are discussed in more detail asfollows. It should be emphasized that, while certain energy biomarkersare discussed and enumerated herein, the invention is not limited tomodulation, normalization or enhancement of only these enumerated energybiomarkers.

Lactic acid (lactate) levels: Mitochondrial dysfunction typicallyresults in abnormal 1 evels of 1 acti c aci d, as pyruvate levels increase and pyruvate i s converted to lactate to maintain capacity forglycolysis. Mitochondrial dysfunction can also result in abnormal levelsof NADH+H+, NADPH+H+, NAD, or NADP, as the reduced nicotinamide adeninedinucleotides are not efficiently processed by the respiratory chain.Lactate levels can be measured by taking samples of appropriate bodilyfluids such as whole blood, plasma, or cerebrospinal fluid. Usingmagnetic resonance, lactate levels can be measured in virtually anyvolume of the body desired, such as the brain.

Measurement of cerebral lactic acidosis using magnetic resonance inMELAS patients is described in Kaufmann et al., Neurology 62(8):1297(2004). Values of the levels of lactic acid in the lateral ventricles ofthe brain are presented for two mutations resulting in MELAS, A3243G andA8344G. Whole blood, plasma, and cerebrospinal fluid lactate levels canbe measured by commercially available equipment such as the YSI 2300STAT Plus Glucose & Lactate Analyzer (YSI Life Sciences, Ohio).

NAD, NADP, NADH and NADPH levels: Measurement of NAD, NADP, NADH(NADH+H+) or NADPH (NADPH+H+) can be measured by a variety offluorescent, enzymatic, or electrochemical techniques, e.g., theelectrochemical assay described in US 2005/0067303.

GSH, GSSG, Cys, and CySS levels: Briefly, plasma levels of GSH, GSSG,Cys, and CySS are used to calculate the in vivo E_(h) values. Samplesare collected using the procedure of Jones et al (2009 Free RadicalBiology & Medicine 47(10) pp 1329-1338), and bromobimane is used toalkylate free thiols and HPLC and either electrochemical or MSMS toseparate, detect, and quantify the molecules. As described in moredetail in PCT Application No. PCT/US2013/058568, a method was developedfor different experimental parameters to analyze the most commonmonothiols and disulfide (cystine, cysteine, reduced (GSH) and oxidizedglutathione (GSSG)) present in human plasma, and usingBathophenanthroline disulfonic acid as the internal standard (IS).Complete separation of all the targets analytes and IS at 35° C. on aC18 RP column (250 mm×4.6 mm, 3 micron) was achieved using 0.2%TFA:Acetonitrile as a mobile phase pumped at the rate of 0.6 ml min-1using electrochemical detector in DC mode at the detector potential of1475 mV.

Oxygen consumption (vO2 or VO2), carbon dioxide output (vCO2 or VCO2),and respiratory quotient (VCO2/VO2): vO2 is usually measured eitherwhile resting (resting vO2) or at maximal exercise intensity (vO2 max).Optimally, both values will be measured. However, for severely disabledpatients, measurement of vO2 max may be impractical. Measurement of bothforms of vO2 is readily accomplished using standard equipment from avariety of vendors, e.g. Korr Medical Technologies, Inc. (Salt LakeCity, Utah). VCO2 can also be readily measured, and the ratio of VCO2 toV02 under the same conditions (VCO2/VO2, either resting or at maximalexercise intensity) provides the respiratory quotient (RQ).

Oxidized Cytochrome C, reduced Cytochrome C, and ratio of oxidizedCytochrome C to reduced Cytochrome C: Cytochrome C parameters, such asoxidized cytochrome C levels (Cyt C_(ox)), reduced cytochrome C levels(Cyt C_(red)), and the ratio of oxidized cytochrome C/reduced cytochromeC ratio (Cyt C_(ox))/(Cyt C_(red)), can be measured by in vivo nearinfrared spectroscopy. See, e.g., Rolfe, P., “In vivo near-infraredspectroscopy,” Annu. Rev. Biomed. Eng. 2:715-54 (2000) and Strangman etal., “Non-invasive neuroimaging using near-infrared light” Biol.Psychiatry 52:679-93 (2002).

Exercise tolerance/Exercise intolerance: Exercise intolerance is definedas “the reduced ability to perform activities that involve dynamicmovement of large skeletal muscles because of symptoms of dyspnea orfatigue” (Pifia et al., Circulation 107:1210 (2003)). Exerciseintolerance is often accompanied by myoglobinuria, due to breakdown ofmuscle tissue and subsequent excretion of muscle myoglobin in the urine.Various measures of exercise intolerance can be used, such as time spentwalking or running on a treadmill before exhaustion, time spent on anexercise bicycle (stationary bicycle) before exhaustion, and the like.Treatment with the compounds, compositions, or methods of the inventioncan result in about a 10% or greater improvement in exercise tolerance(for example, about a 10% or greater increase in time to exhaustion,e.g. from 10 minutes to 11 minutes), about a 20% or greater improvementin exercise tolerance, about a 30% or greater improvement in exercisetolerance, about a 40% or greater improvement in exercise tolerance,about a 50% or greater improvement in exercise tolerance, about a 75% orgreater improvement in exercise tolerance, or about a 100% or greaterimprovement in exercise tolerance. While exercise tolerance is not,strictly speaking, an energy biomarker, for the purposes of theinvention, modulation, normalization, or enhancement of energybiomarkers includes modulation, normalization, or enhancement ofexercise tolerance.

Similarly, tests for normal and abnormal values of pyruvic acid(pyruvate) levels, lactate/pyruvate ratio, ATP levels, anaerobicthreshold, reduced coenzyme Q (CoQ_(red)) levels, oxidized coenzyme Q(CoQ_(ox)) levels, total coenzyme Q (CoQ_(tot)) levels, oxidizedcytochrome C levels, reduced cytochrome C levels, oxidized cytochromeC/reduced cytochrome C ratio, GSH and cysteine reduced, oxidized, totallevels and ratio, acetoacetate levels, β-hydroxy butyrate levels,acetoacetate/β-hydroxy butyrate ratio, 8-hydroxy-2′-deoxyguanosine(8-OHdG) levels, and levels of reactive oxygen species are known in theart and can be used to evaluate efficacy of the compounds, compositions,and methods of the invention. (For the purposes of the invention,modulation, normalization, or enhancement of energy biomarkers includesmodulation, normalization, or enhancement of anaerobic threshold.)

Table 8, following, illustrates the effect that various dysfunctions canhave on biochemistry and energy biomarkers. It also indicates thephysical effect (such as a disease symptom or other effect of thedysfunction) typically associated with a given dysfunction. It should benoted that any of the energy biomarkers listed in the table, in additionto energy biomarkers enumerated elsewhere, can also be modulated,enhanced, or normalized by the compounds, compositions, and methods ofthe invention. RQ=respiratory quotient; BMR=basal metabolic rate; HR(CO)=heart rate (cardiac output); T=body temperature (preferablymeasured as core temperature); AT=anaerobic threshold; pH=blood pH(venous and/or arterial).

TABLE 8 Site of Biochemical Measurable Energy Physical Dysfunction EventBiomarker Effect Respiratory ↑ NADH Δ lactate, Metabolic Chain Δlactate: pyruvate dyscrasia & ratio; and fatigue Δ acetoacetate:β-hydroxy butyrate ratio Respiratory ↓ H+ gradient Δ ATP Organ dependentChain dysfunction Respiratory ↓ Electron flux Δ VO2, RQ, BMR, MetabolicChain ΔT, AT, pH dyscrasia & fatigue Mitochondria & ↓ ATP, ↓ VO2 Δ Work,ΔHR (CO) Exercise cytosol intolerance Mitochondria & ↓ ATP Δ PCrExercise cytosol intolerance Respiratory ↓ Cyt Co_(x)/_(Red) Δ λ~700-900 nm (Near Exercise Chain Infrared Spectroscopy) intoleranceIntermediary ↓ Catabolism Δ Cl4-Labeled substrates Metabolic metabolismdyscrasia & fatigue Respiratory ↓ Electron flux Δ Mixed Venous VO2Metabolic Chain dyscrasia & fatigue Mitochondria & ↑ Oxidative stress ΔTocopherol & Uncertain cytosol Tocotrienols, CoQ10, docosahexaenoic acidMitochondria & ↑ Oxidative stress Δ Glutathionered Uncertain cytosolMitochondria & Nucleic acid Δ8-hydroxy 2-deoxy Uncertain cytosoloxidation guanosme Mitochondria & Lipid oxidation Δ Isoprostane(s),Uncertain cytosol eicosanoids Cell membranes Lipid oxidation Δ Ethane(breath) Uncertain Cell membranes Lipid oxidation Δ MalondialdehydeUncertain

Treatment of a subject afflicted by an oxidative stress disorder inaccordance with the methods of the invention may result in theinducement of a reduction or alleviation of symptoms in the subject,e.g., to halt the further progression of the disorder.

Partial or complete suppression of the oxidative stress disorder canresult in a lessening of the severity of one or more of the symptomsthat the subject would otherwise experience. For example, partialsuppression of MELAS could result in reduction in the number ofstroke-like or seizure episodes suffered.

Any one or any combination of the energy biomarkers described hereinprovide conveniently measurable benchmarks by which to gauge theeffectiveness of treatment or suppressive therapy. Additionally, otherenergy biomarkers are known to those skilled in the art and can bemonitored to evaluate the efficacy of treatment or suppressive therapy.

Use of Compounds or Compositions for Modulation of Energy Biomarkers

In addition to monitoring energy biomarkers to assess the status oftreatment or suppression of oxidative stress diseases, the compounds orcompositions of the invention can be used in subjects or patients tomodulate one or more energy biomarkers. Modulation of energy biomarkerscan be done to normalize energy biomarkers in a subject, or to enhanceenergy biomarkers in a subject.

Normalization of one or more energy biomarkers is defined as eitherrestoring the level of one or more such energy biomarkers to normal ornear-normal levels in a subject whose levels of one or more energybiomarkers show pathological differences from normal levels (i.e.,levels in a healthy subject), or to change the levels of one or moreenergy biomarkers to alleviate pathological symptoms in a subject.Depending on the nature of the energy biomarker, such levels may showmeasured values either above or below a normal value. For example, apathological lactate level is typically higher than the lactate level ina normal (i.e., healthy) person, and a decrease in the level may bedesirable. A pathological ATP level is typically lower than the ATPlevel in a normal (i.e., healthy) person, and an increase in the levelof ATP may be desirable. Accordingly, normalization of energy biomarkerscan involve restoring the level of energy biomarkers to within about atleast two standard deviations of normal in a subject, more preferably towithin about at least one standard deviation of normal in a subject, towithin about at least one-half standard deviation of normal, or towithin about at least one-quarter standard deviation of normal.

Enhancement of the level of one or more energy biomarkers is defined aschanging the extant levels of one or more energy biomarkers in a subjectto a level which provides beneficial or desired effects for the subject.For example, a person undergoing strenuous effort or prolonged vigorousphysical activity, such as mountain climbing, could benefit fromincreased ATP levels or decreased lactate levels. As described above,normalization of energy biomarkers may not achieve the optimum state fora subject with an oxidative stress disease, and such subjects can alsobenefit from enhancement of energy biomarkers. Examples of subjects whocould benefit from enhanced levels of one or more energy biomarkersinclude, but are not limited to, subjects undergoing strenuous orprolonged physical activity, subjects with chronic energy problems, orsubjects with chronic respiratory problems. Such subjects include, butare not limited to, pregnant females, particularly pregnant females inlabor; neonates, particularly premature neonates; subjects exposed toextreme environments, such as hot environments (temperatures routinelyexceeding about 85-86 degrees Fahrenheit or about 30 degrees Celsius forabout 4 hours daily or more), cold environments (temperatures routinelybelow about 32 degrees Fahrenheit or about 0 degrees Celsius for about 4hours daily or more), or environments with lower-than-average oxygencontent, higher-than-average carbon dioxide content, orhigher-than-average levels of air pollution (airline travelers, flightattendants, subjects at elevated altitudes, subjects living in citieswith lower-than-average air quality, subjects working in enclosedenvironments where air quality is degraded); subjects with lung diseasesor lower-than-average lung capacity, such as tubercular patients, lungcancer patients, emphysema patients, and cystic fibrosis patients;subjects recovering from surgery or illness; elderly subjects, includingelderly subjects experiencing decreased energy; subjects suffering fromchronic fatigue, including chronic fatigue syndrome; subjects undergoingacute trauma; subjects in shock; subjects requiring acute oxygenadministration; subjects requiring chronic oxygen administration; orother subjects with acute, chronic, or ongoing energy demands who canbenefit from enhancement of energy biomarkers.

Accordingly, when an increase in a level of one or more energybiomarkers is beneficial to a subject, enhancement of the one or moreenergy biomarkers can involve increasing the level of the respectiveenergy biomarker or energy biomarkers to about at least one-quarterstandard deviation above normal, about at least one-half standarddeviation above normal, about at least one standard deviation abovenormal, or about at least two standard deviations above normal.Alternatively, the level of the one or more energy biomarkers can beincreased by about at least 10% above the subject's level of therespective one or more energy biomarkers before enhancement, by about atleast 20% above the subject's level of the respective one or more energybiomarkers before enhancement, by about at least 30% above the subject'slevel of the respective one or more energy biomarkers beforeenhancement, by about at least 40% above the subject's level of therespective one or more energy biomarkers before enhancement, by about atleast 50% above the subject's level of the respective one or more energybiomarkers before enhancement, by about at least 75% above the subject'slevel of the respective one or more energy biomarkers beforeenhancement, or by about at least 100% above the subject's level of therespective one or more energy biomarkers before enhancement.

When a decrease in a level of one or more energy biomarkers is desiredto enhance one or more energy biomarkers, the level of the one or moreenergy biomarkers can be decreased by an amount of about at leastone-quarter standard deviation of normal in a subject, decreased byabout at least one-half standard deviation of normal in a subject,decreased by about at least one standard deviation of normal in asubject, or decreased by about at least two standard deviations ofnormal in a subject. Alternatively, the level of the one or more energybiomarkers can be decreased by about at least 10% below the subject'slevel of the respective one or more energy biomarkers beforeenhancement, by about at least 20% below the subject's level of therespective one or more energy biomarkers before enhancement, by about atleast 30% below the subject's level of the respective one or more energybiomarkers before enhancement, by about at least 40% below the subject'slevel of the respective one or more energy biomarkers beforeenhancement, by about at least 50% below the subject's level of therespective one or more energy biomarkers before enhancement, by about atleast 75% below the subject's level of the respective one or more energybiomarkers before enhancement, or by about at least 90% below thesubject's level of the respective one or more energy biomarkers beforeenhancement.

Use of Compounds or Compositions in Research Applications, ExperimentalSystems, and Assays

The compounds or compositions of the invention can also be used inresearch applications. They can be used in in vitro, in vivo, or ex vivoexperiments to modulate one or more energy biomarkers in an experimentalsystem. Such experimental systems can be cell samples, tissue samples,cell components or mixtures of cell components, partial organs, wholeorgans, or organisms. Any one or more of the compounds or compositionscan be used in experimental systems or research applications. Suchresearch applications can include, but are not limited to, use as assayreagents, elucidation of biochemical pathways, or evaluation of theeffects of other agents on the metabolic state of the experimentalsystem in the presence/absence of one or more compounds or compositionsof the invention.

Additionally, the compounds or compositions of the invention can be usedin biochemical tests or assays. Such tests can include incubation of oneor more compounds or compositions of the invention with a tissue or cellsample from a subject to evaluate a subject's potential response (or theresponse of a specific subset of subjects) to administration of said oneor more compounds or compositions, or to determine which compound orcomposition of the invention produces the optimum effect in a specificsubject or subset of subjects. One such test or assay would involve 1)obtaining a cell sample or tissue sample from a subject in whichmodulation of one or more energy biomarkers can be assayed; 2)administering one or more compounds or compositions of the invention tothe cell sample or tissue sample; and 3) determining the amount ofmodulation of the one or more energy biomarkers after administration ofthe one or more compounds or compositions, compared to the status of theenergy biomarker prior to administration of the one or more compounds orcompositions. Another such test or assay would involve 1) obtaining acell sample or tissue sample from a subject in which modulation of oneor more energy biomarkers can be assayed; 2) administering at least twocompounds or compositions of the invention to the cell sample or tissuesample; 3) determining the amount of modulation of the one or moreenergy biomarkers after administration of the at least two compounds orcompositions, compared to the status of the energy biomarker prior toadministration of the at least two compounds or compositions, and 4)selecting a compound or composition for use in treatment, suppression,or modulation based on the amount of modulation determined in step 3.

In certain embodiments, provided herein are methods for the use of apolymorph of Form I-VI for treating or protecting against injury ordamage caused by radiation exposure and methods of using such compoundsfor treating or for protecting against injury or damage caused byradiation exposure. In certain embodiments, the methods for treating orfor protecting against injury or damage caused by radiation exposure,comprise administering to a cell or cells, a tissue or tissues, or asubject in need thereof, a therapeutically effective amount or aprophylactically effective amount of a polymorph of Form I-VI orcomposition disclosed herein. In one embodiment, the polymorph of FormI-VI are used therapeutically during, after, or during and afterradiation exposure. In another embodiment, a polymorph of Form I-VI areused prophylactically prior to radiation exposure. In anotherembodiment, a polymorph of Form I-VI is administered concurrently withradiation exposure. In another embodiment, the one or more compounds areadministered after radiation exposure.

In certain embodiments, provided herein are methods for the use of apolymorph of Form I-VI for treating against injury or damage caused byradiation exposure and methods of using such compounds for treating orfor protecting against injury or damage caused by radiation exposure. Incertain embodiments, the methods for treating an injury or damage causedby radiation exposure, comprise administering to a cell or cells, atissue or tissues, or a subject in need thereof, a therapeuticallyeffective amount or a prophylactically effective amount of a polymorphof Form I-VI or composition disclosed herein. In one embodiment, thepolymorph of Form I-VI are used therapeutically during, after, or duringand after radiation exposure. In another embodiment, a polymorph of FormI-VI are used prophylactically prior to radiation exposure. In anotherembodiment, a polymorph of Form I-VI is administered concurrently withradiation exposure. In another embodiment, the one or more compounds areadministered after radiation exposure.

Pharmaceutical Formulations

The compounds or compositions described herein can be formulated aspharmaceutical compositions by formulation with additives such aspharmaceutically acceptable excipients, pharmaceutically acceptablecarriers, and pharmaceutically acceptable vehicles. Suitablepharmaceutically acceptable excipients, carriers and vehicles includeprocessing agents and drug delivery modifiers and enhancers, such as,for example, calcium phosphate, magnesium stearate, talc,monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose,hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, low melting waxes,ion exchange resins, and the like, as well as combinations of any two ormore thereof. Other suitable pharmaceutically acceptable excipients aredescribed in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., NewJersey (1991), and “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and21st edition (2005), incorporated herein by reference.

A pharmaceutical composition can comprise a unit dose formulation, wherethe unit dose is a dose sufficient to have a therapeutic or suppressiveeffect or an amount effective to modulate, normalize, or enhance anenergy biomarker. The unit dose may be sufficient as a single dose tohave a therapeutic or suppressive effect or an amount effective tomodulate, normalize, or enhance an energy biomarker. Alternatively, theunit dose may be a dose administered periodically in a course oftreatment or suppression of a disorder, or to modulate, normalize, orenhance an energy biomarker.

Pharmaceutical compositions containing the compounds or compositions ofthe invention may be in any form suitable for the intended method ofadministration, including, for example, a solution, a suspension, or anemulsion. Liquid carriers are typically used in preparing solutions,suspensions, and emulsions. Liquid carriers contemplated for use in thepractice of the present invention include, for example, water, saline,pharmaceutically acceptable organic solvent(s), pharmaceuticallyacceptable oils or fats, and the like, as well as mixtures of two ormore thereof. The liquid carrier may contain other suitablepharmaceutically acceptable additives such as solubilizers, emulsifiers,nutrients, buffers, preservatives, suspending agents, thickening agents,viscosity regulators, stabilizers, and the like. Suitable organicsolvents include, for example, monohydric alcohols, such as ethanol, andpolyhydric alcohols, such as glycols. Suitable oils include, forexample, soybean oil, coconut oil, olive oil, safflower oil, cottonseedoil, and the like. For parenteral administration, the carrier can alsobe an oily ester such as ethyl oleate, isopropyl myristate, and thelike. Compositions of the present invention may also be in the form ofmicroparticles, microcapsules, liposomal encapsulates, and the like, aswell as combinations of any two or more thereof.

Time-release or controlled release delivery systems may be used, such asa diffusion controlled matrix system or an erodible system, as describedfor example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise onControlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., NewYork 1992. The matrix may be, for example, a biodegradable material thatcan degrade spontaneously in situ and in vivo for, example, byhydrolysis or enzymatic cleavage, e.g., by proteases. The deliverysystem may be, for example, a naturally occurring or synthetic polymeror copolymer, for example in the form of a hydrogel. Exemplary polymerswith cleavable linkages include polyesters, polyorthoesters,polyanhydrides, polysaccharides, poly(phosphoesters), polyamides,polyurethanes, poly(imidocarbonates) and poly(phosphazenes).

The compounds or compositions of the invention may be administeredenterally, orally, parenterally, sublingually, by inhalation (e.g. asmists or sprays), rectally, or topically in dosage unit formulationscontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired. For example, suitable modes ofadministration include oral, subcutaneous, transdermal, transmucosal,iontophoretic, intravenous, intraarterial, intramuscular,intraperitoneal, intranasal (e.g. via nasal mucosa), subdural, rectal,gastrointestinal, and the like, and directly to a specific or affectedorgan or tissue. For delivery to the central nervous system, spinal andepidural administration, or administration to cerebral ventricles, canbe used. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques. The compounds or compositions are mixed withpharmaceutically acceptable carriers, adjuvants, and vehiclesappropriate for the desired route of administration. Oral administrationis a preferred route of administration, and formulations suitable fororal administration are preferred formulations. The compounds describedfor use herein can be administered in solid form, in liquid form, inaerosol form, or in the form of tablets, pills, powder mixtures,capsules, granules, injectables, creams, solutions, suppositories,enemas, colonic irrigations, emulsions, dispersions, food premixes, andin other suitable forms. The compounds or compositions can also beadministered in liposome formulations. The compounds can also beadministered as prodrugs, where the prodrug undergoes transformation inthe treated subject to a form which is therapeutically effective.Additional methods of administration are known in the art.

In some embodiments of the invention, especially those embodiments wherea formulation is used for injection or other parenteral administrationincluding the routes listed herein, but also including embodiments usedfor oral, gastric, gastrointestinal, or enteric administration, theformulations and preparations used in the methods of the invention aresterile. Sterile pharmaceutical formulations are compounded ormanufactured according to pharmaceutical-grade sterilization standards(United States Pharmacopeia Chapters 797, 1072, and 1211; CaliforniaBusiness & Professions Code 4127.7; 16 California Code of Regulations1751, 21 Code of Federal Regulations 211) known to those of skill in theart.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in propylene glycol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also compriseadditional substances other than inert diluents, e.g., lubricatingagents such as magnesium stearate. In the case of capsules, tablets, andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

The compounds or compositions of the present invention can also beadministered in the form of liposomes. As is known in the art, liposomesare generally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multilamellar hydrated liquid crystalsthat are dispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p.33 et seq (1976).

The invention also provides articles of manufacture and kits containingmaterials useful for treating or suppressing oxidative stress disorders.The invention also provides kits comprising any one or more of thecompounds or compositions as described herein. In some embodiments, thekit of the invention comprises a suitable container.

In other aspects, the kits may be used for any of the methods describedherein, including, for example, to treat an individual with amitochondrial disorder, or to suppress a mitochondrial disorder in anindividual.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost to which the active ingredient is administered and the particularmode of administration. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, body area, body mass index (BMI),general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the type,progression, and severity of the particular disease undergoing therapy.The pharmaceutical unit dosage chosen is usually fabricated andadministered to provide a defined final concentration of drug in theblood, tissues, organs, or other targeted region of the body. Thetherapeutically effective amount or effective amount for a givensituation can be readily determined by routine experimentation and iswithin the skill and judgment of the ordinary clinician.

Examples of dosages which can be used are a therapeutically effectiveamount or effective amount within the dosage range of about 0.1 mg/kg toabout 300 mg/kg body weight, or within about 1.0 mg/kg to about 100mg/kg body weight, or within about 1.0 mg/kg to about 50 mg/kg bodyweight, or within about 1.0 mg/kg to about 30 mg/kg body weight, orwithin about 1.0 mg/kg to about 10 mg/kg body weight, or within about 10mg/kg to about 100 mg/kg body weight, or within about 50 mg/kg to about150 mg/kg body weight, or within about 100 mg/kg to about 200 mg/kg bodyweight, or within about 150 mg/kg to about 250 mg/kg body weight, orwithin about 200 mg/kg to about 300 mg/kg body weight, or within about250 mg/kg to about 300 mg/kg body weight. Compounds or compositions ofthe present invention may be administered in a single daily dose, or thetotal daily dosage may be administered in divided dosage of two, threeor four times daily.

While the compounds or compositions of the invention can be administeredas the sole active pharmaceutical agent, they can also be used incombination with one or more other agents used in the treatment orsuppression of disorders. Representative agents useful in combinationwith the compounds or compositions of the invention for the treatment orsuppression of mitochondrial diseases include, but are not limited to,Coenzyme Q, vitamin E, idebenone, MitoQ, vitamins, NAC, and antioxidantcompounds.

When additional active agents are used in combination with the compoundsor compositions of the present invention, the additional active agentsmay generally be employed in therapeutic amounts as indicated in thePhysicians' Desk Reference (PDR) 53rd Edition (1999), or suchtherapeutically useful amounts as would be known to one of ordinaryskill in the art.

The compounds or compositions of the invention and the othertherapeutically active agents can be administered at the recommendedmaximum clinical dosage or at lower doses. Dosage levels of the activecompounds in the compositions of the invention may be varied so as toobtain a desired therapeutic response depending on the route ofadministration, severity of the disease and the response of the patient.When administered in combination with other therapeutic agents, thetherapeutic agents can be formulated as separate compositions that aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

The invention will be further understood by the following nonlimitingexamples.

Preparation of Compositions of the Invention

The compositions of this invention can be prepared from readilyavailable starting materials using the following general methods andprocedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Synthetic Reaction Parameters

Solvents employed in synthesis of the compounds and compositions of theinvention include, for example, methanol (“MeOH”), acetone, water,acetonitrile, 1,4-dioxane, dimethylformamide (“DMF”), benzene, toluene,xylene, tetrahydrofuran (“THF”), chloroform, methylene chloride (ordichloromethane, (“DCM”)), diethyl ether, pyridine,2-methyl-tetrahydrofuran (“2-MeTHF”), dimethylacetamide (“DMA”), ethylacetate (“EtOAc”), ethanol (“EtOH”), isopropyl alcohol (“IPA”),isopropyl acetate (“IPAc”), methyl cellulose (“MC”), acetonitrile(“MeCN”), methanol (MeOH), methyl tert-butyl ether (“MTBE”), phosphatebuffered saline (“PBS”), tetrahydrofuran (“THF”), and the like, as wellas mixtures thereof.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

The compounds and compositions herein are synthesized by an appropriatecombination of generally well-known synthetic methods. Techniques usefulin synthesizing the compounds and compositions herein are both readilyapparent and accessible to those of skill in the relevant art in lightof the teachings described herein. The discussion below is offered toillustrate certain of the diverse methods available for use inassembling the compounds and compositions herein. However, thediscussion is not intended to define the scope of reactions or reactionsequences that are useful in preparing the compounds and compositionsherein.

Other methods for producing the compounds and compositions of theinvention will be apparent to one skilled in the art in view of theteachings herein.

EXAMPLES Example 1. Synthesis of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide(Form I) Example 1A. Extraction of (1 S, 2S)-(+)-Pseudoephedrine FreeBase

To a suspension of (1S, 2S)-(+)-Pseudoephedrine hydrochloride salt (300g, Spectrum) in 2-MeTHF (1.5 L, 5 vol) was added 20% Aq NaOH solution(750 mL, 2.5 vol) and the mixture was stirred for 30 min (some solidsremained undissolved) and transferred to a separatory funnel. The loweraqueous layer was drained along with solids that remained at theinterphase and back extracted with 2-MeTHF (750 mL, 2.5 vol), theundissolved solids completely dissolved to form two clear layers. Thecombined organic layers were evaporated to dryness on rotavapor and thesolids obtained were dried in a vacuum oven at 50° C. overnight toafford 240.3 g of free base as a white solid (97.7% recovery).

Example 1B. Precipitation of (1S,2S)-Pseudoephedrine from2-MeTHF/heptane

(1S,2S)-pseudoephedrine (Sigma-Aldrich, sku #212464, 8.2 g) wasdissolved at 50° C. in 2-MeTHF (41 ml, 5 vol). The resulting solutionwas diluted with heptane (82 ml, 10 vol) and the resulting suspensionwas stirred at room temperature overnight. The crystallized(1S,2S)-pseudoephedrine was filtered off and dried overnight at 40°under vacuum affording 6.4 g (78%) of white crystalline material.Filtrate was discarded to general waste.

Relatively low (78%) crystallization yield prompted an additionalcrystallization experiment with higher heptane to 2-MeTHF ratio.Crystalline (1S,2S)-pseudoephedrine obtained in the experiment above wasdissolved at 50° C. in 2-MeTHF (32 ml, 5 vol). The resulting solutionwas diluted with heptane (32 ml, 5 vol) and the resulting suspension waschased with heptane (3×50 ml) on rotary evaporator until molar ration of2-MeTHF to heptane became lower than 6% by NMR. The resulting suspensionwas filtered off and the product dried overnight at 40° under vacuumaffording 6.3 g (98%) of white crystalline material.

Example 1C. Chiral Resolution of Trolox Using (1S,2S)-(+)-Pseudoephedrine

Racemic Trolox (316.6 g, 1.27 mol) and (1S, 2S)-(+)-Pseudoephedrine freebase described in Example 1A (240.0 g, 1.46 mol) were charged to a 4 Ljacketed reactor equipped with an overhead stirrer, temperature probeand a nitrogen purge. Ethyl acetate (EtOAc, 1585 mL, 5 vol) was chargedand the slurry was heated to 50° C. resulting in clear solution.(Premature (prior to complete dissolution of rac-trolox) precipitationof the (R)-trolox-pseudoephedrine salt was occasionally observed at 40°.If premature precipitation takes place the reaction mixture was heated(usually to reflux temperature) to achieve complete dissolution.) Thereaction mixture was cooled overnight to room temperature at which timemassive precipitation was observed. The mixture was cooled to 10° C.over 30 min and held at this temperature for 1 h. The solids formed werecollected by filtration, the wet cake was washed with EtOAc (1.9 L, 6Vol) and the filter cake was dried in a vacuum oven at 25-30° C. toconstant weight to afford 188.1 g (71.3% based on (R)-trolox) of a whitesolid. Chiral HPLC data indicated nearly 100% enantiomeric purity.

Example 1D. Recovery of (R)-Trolox from its Salt with (1S,2S)-(+)-Pseudoephedrine

The resulting (R)-Trolox PE salt (187.3 g, 0.45 mol) was charged to a 2L round-bottom flask followed by 2-MeTHF (570 ml, 3 vol.) to form aslurry. Hydrochloric acid (2.5 M, 325 ml, 0.8 mol, 1.75 eq) was addedportionwise while maintaining temperature below 25° C. The trolox-PEsalt was dissolved and (R)-Trolox was extracted into organic phase.Small black rag was observed in the interface and was kept with theaqueous. The aqueous phase was additionally extracted with 2-MeTHF(2×200 ml). The combined organic layer was then washed with 15% NaCl(200 ml) followed by water (200 ml). The organic layer was dried overanhydrous sodium sulfate (150 g), filtered and evaporated to dryness toafford white solid which was dried under vacuum oven at 30° C. toconstant weight of 128.3 g, which is an overstoichiometric amount.

Example 1E. Preparation of(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide

CDI (Sigma-Aldrich) (188 g, 1.16 mol) was charged to a 3-neck 2 L RBFequipped with an overhead stirrer, nitrogen inlet and temperature probe.2-MeTHF (290 mL) was added to give a stirrable slurry followed by slowaddition of (R)-trolox (126.0 g, 504 mmol) in 2-MeTHF (500 ml) at below30° C. A slightly exothermic reaction accompanied by CO₂ evolution wasobserved. Outgassing started after addition of approximately one thirdof (R)-trolox. Complete dissolution of the starting materials wasobserved in approximately 15 min.

The content of this flask was slowly added to a pre-cooled to 5° C.28-30% aqueous ammonia (380 ml) maintaining temperature below 30° C. Theresulting biphasic suspension was stirred at room temperature andmonitored by HPLC. The reaction was found to be complete at 36 h and wasfurther processed after 48 h.

The reaction mixture was acidified to pH 1-2 with sulfuric acid (1:4v/v) (850 ml) maintaining the temperature ≤28° C., reaction was highlyexothermic. The aqueous layer (pH=1) was removed and the organic layerwas washed with NaCl (15% aqueous w/v, 250 mL), NaHCO₃ (1 M, 250 mL),NaCi (15% aqueous w/v, 250 mL) and water (250 ml). The majority of theorganic layer was used for the subsequent steps.

Example 1F. Preparation of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide

A solution of (R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide(708 ml) which contains˜0.39 mole of the intermediate amide and water(126 ml) were charged to a 2 L 3N RBF equipped with an overhead stirrerand a thermocouple.

A stock solution of FeCl₃×6H₂O (480 g, 1.78 mol) in water (336 ml) wasdivided into 4 equal parts (204 g each) and one-fourth of the iron(III)chloride solution was added to the reaction flask. A weak (˜3° C.)exotherm was observed, the color of the organic layer turned nearlyblack then lightened to dark-brown. The biphasic reaction mixture wasvigorously stirred for 40 min at room temperature. After removal of thelightly colored aqueous phase another portion of the iron(III) chloridesolution was added and stirred for 40 min. The operation was repeatedone more time and the organic phase was stored overnight at roomtemperature. The fourth treatment with FeCl₃×6H₂O was performed nextmorning. Nearly complete (99.44%) conversion of(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide to(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidewas observed. Initial iron extraction was performed with 1M trisodiumcitrate solution (2×350 ml); the AUC % of(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide increased to0.84%. pH of the organic phase remained highly acidic (pH=1). A Imlaliquot of the organic phase was treated with 1M NaHCO₃ resulting inmassive precipitation of red Fe(OH)₃. Based on this observation one moretrisodium citrate wash (175 ml) was performed (0.74%(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide). The repeattesting of the 1 ml aliquot with 1M NaHCO₃ gave no precipitation in theaqueous layer and the color of the aqueous layer was yellow, not red,indicating complete or nearly complete iron removal.

The organic layer was heated to 40° C. to prevent prematureprecipitation of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand washed with 1M sodium bicarbonate solution (175 ml). The phase splitwas not immediate but was complete in 15 min forming two clear yellowlayers. The organic layer (0.30%(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide) wasadditionally washed with water (350 ml) giving 0.22%(R)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxamide. Evaporation ofthe organic layer gave 96 g of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

The combined bicarbonate/water layers were back extracted with 2×250 mlof 2-MeTHF. Evaporation of these extracts separately gave 4.0 and 0.9 gof(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

The combined solids (100.9 g—crude(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,84% yield based on (R)-trolox-pseudoephedrine salt) were dissolved inisopropanol (600 ml) at 70° C. and the resulting yellow solution wascharged to a 2 L 3N RBF equipped with an overhead stirrer a heatingmantle and a thermocouple.

Heptane (600 ml) was added, no precipitation was observed. The reactionmixture was reheated to 55° C. and slowly cooled down to roomtemperature. Seeds of the desired polymorph (0.2 g) were added and thereaction mixture was stirred overnight at room temperature. Massiveprecipitation was observed overnight. The reaction mixture was cooled to7° C. and stirred for additional 8 hours. The product was filtered,washed with isopropanol-heptane 1:1 v/v (2×75 ml) and dried over theweekend at 40° C. Yield 69.4 g of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide(58% based on (R)-trolox—pseudoephedrine salt). XRPD data for theproduct corresponded to the desired Form I.

Example 2. Solubility Measurement of Pattern A

(R)-Trolox was produced from racemic Trolox via methylbenzyl aminedouble resolution in a manner similar to that described in Example 29 ofU.S. Pat. No. 4,026,907. This (R)-Trolox was used to synthesize the(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidestarting material. This starting material, designated as Pattern A, wasused for the solubility measurement.

Excess amount of solid was slurried in 17 solvent systems having diverseproperties for minimum of 3 days. The slurry was centrifuged and theclear solution was used for gravimetric method. The compound showedelevated solubility in MeOH, EtOH at ambient temperature, and IPA,acetone, MeOH, EtOH and 2-MeTHF at 50° C. Moderate solubility wasobserved in EtOAc, THF, IPA, acetone, 2-MeTHF, MeCN, 0.5% MethylCellulose/2% Tween 80, IPAc and 4% DMA in PBS at ambient and 50° C.Limited solubility was observed from heptane, toluene, MTBE, water and0.5% Methyl Cellulose in water at ambient and elevated temperatures.Table 9 presents the measured solubility data. A ±10% error is expected.

TABLE 9 Solubility of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide in various solvent systems(Starting material, Pattern A used) mg/mL at mg/mL at Solvent 25° C. 50°C. Heptane 6 6 Toluene 3 <18 MTBE 10 16 EtOAc 22 38 THF 28. 33 IPA 20 53Acetone 39 90 EtOH 40 >121 MeOH >101 >112 2-MeTHF 35 59 MeCN 20 38 Water3 3 ¹Water 2 6 0.5% MC/2% Tween 26 30 80* IPAc 13 16 0.5% MC in Water* 74 4% DMA in PBS* 10 13 *Part of the concentration relates to solventconstituents

As shown in Table 9, Pattern A is soluble in a variety of polar andnon-polar solvents, and in addition has increased solubility in a simpledetergent (0.5% MC/2% Tween 80).

Example 3. Short Term Slurry Experiments

Short term slurry experiments of the starting material, Pattern A, wereperformed for a minimum of 3 days in 17 different solvent systems havingdiverse properties at two different temperatures (25 and 50° C.). A 48position Chemglass reaction block was used for heating and stirring theslurries which were in 2 mL HPLC vials. After the due time, vials werecentrifuged and the wet solids were used for X-ray diffraction. Table 10shows the results of the slurry experiments. These results demonstratethat Pattern A solid was relatively stable if slurried in most of thesesolvents for a short period of time. However, slurring Pattern A solidin THF and 0.5% Methyl Cellulose/2% Tween 80 resulted in two new X-raypatterns designated as Patterns B and C respectively (FIG. 1).

TABLE 10 Summary of a minimum 3 days slurry of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide(Starting material, Pattern A used) 25° C. 50° C. Starting ResultingResulting Resulting Solvent Form Form, Wet Form, Dry Form, Wet Heptane AA A Toluene A A A MTBE A A A EtOAc A A A THF A B B - IPA A A A Acetone AA A EtOH A A - MeOH A - - 2-MeTHF A A A MeCN A A A Water A A A ¹Water AA A 0.5% MC/2% Tween 80 A C C A IPAc A A A 0.5% MC in Water A A A 4% DMAin PBS A A A

Example 4. Evaporative Crystallization Experiments

Evaporative crystallization experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidewere performed using the samples generated during the gravimetricsolubility determination (Example 2). XRPD analysis of most samplesafforded Pattern A. However XRPD analysis of solids isolated from2-MeTHF at 25° C. was found to afford a unique crystalline pattern,designated Pattern D as shown in FIG. 2. XRPD analysis of samples fromIPAc, 0.5% MC in water and 4% DMA in PBS mostly yielded amorphouspatterns with the exception of IPAc at 50° C. which afforded acrystalline pattern consistent with Pattern A. All results aresummarized in Table 11.

TABLE 11 Summary of evaporative crystallization experiments of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide (Starting material, Pattern A used) 25° C. 50° C.Starting Resulting Resulting Resulting Solvent Form Form, Wet Form, DryForm, Wet Heptane A — — Toluene A A A MTBE A Oil Amorphous EtOAc A A ATHF A Amorphous A IPA A Amorphous Amorphous Acetone A A A EtOH A A AMeOH A A A 2-MeTHF A D NA A MeCN A A A Water A A A ¹ Water A A A 0.5%MC/2% Tween A Oil Oil 80 IPAc A Amorphous A 0.5% MC in Water A Amorphous— 4% DMA in PBS A Amorphous Amorphous NA—Drying not performed due tosample dried overnight

Example 5. Crystallization Experiments

Fast and slow cooling single solvent crystallization experiments wereperformed in Toluene, EtOAc, IPA, acetone, EtOH, 2-MeTHF, and IPA with2% water (Table 12). A 48 position Chemglass reaction block was used forheating and stirring which were performed in 4 mL vials. Each vial wascharged with 50-80 mg of starting material (Pattern A) fitted with amagnetic stir bar. Primary solvent was added and heated with stirringuntil dissolution achieved. Once fully dissolved the sample was slowcooled by radiative cooling or crashed cooled with use of an ice bathfollowed over night equilibration with stirring. For Binary solventcrystallizations, anti-solvent (Heptane) was added in two methods (Table13). In method one, the anti-solvent was added drop wise to the samplesolution until slight precipitation was observed. Method two used areverse addition of the sample solution to a heated anti-solvent in a2:1 ratio before being allowed to cool. Samples which afforded solidsafter overnight equilibration were isolated by filtration and samplesthat did not precipitate were evaporated under a gentle stream ofnitrogen. All samples were dried overnight in a vacuum oven at ambientconditions and analyzed by XRPD to check for form change. Allexperimental details and results are summarized in Tables 12-13.

XRPD analysis of all isolated solids mostly afforded crystallinepatterns consistent with the starting material, Pattern A. However,single solvent crystallizations performed in 2-MeTHF with fast and slowcooling profiles, were not observed to afford solids upon cooling andwere evaporated to dryness under nitrogen. These evaporated solids werefound to afford XRPD patterns consistent with previously observedPattern D (FIG. 3). Fast cooling crystallization performed in EtOAc,yielded unique crystalline solids by XRPD which were compared to allknown forms and designated as Pattern E (FIG. 4). Binary solventcrystallizations with fast and slow cooling profiles performed inEtOH/Heptane and Acetone/Heptane afforded XRPD patterns consistent withPattern A.

TABLE 12 Summary of single solvent crystallization experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide (Startingmaterial, Pattern A used) Starting Material (Pattern Primary Solvent A)Vol Temp Cooling (mg) Solvent (mL) (C) Rate Isolation XRPD [Pattern]49.7 Toluene 2.75 60 Fast NA — 52.3 EtOAc 1 60 Fast Filter Crystalline[Pattern E] 55.9 IPA 1 60 Fast Filter Crystalline [Pattern A] 50.9Acetone 0.5 60 Fast Filter Crystalline [Pattern A] 82.0 EtOH 0.5 60 FastFilter Crystalline [Pattern A] 51.1 2-MeTHF 1 60 Fast Evap Crystalline[Pattern D] 48.9 IPA with 0.5 60 Fast Filter Crystalline [Pattern A] 2%water 50.2 Toluene 2.75 60 Slow NA — 49.2 EtOAc 1 60 Slow FilterCrystalline [Pattern A] 54.9 IPA 1 60 Slow Filter Crystalline [PatternA] 51.9 Acetone 0.5 60 Slow Filter Crystalline [Pattern A] 82.7 EtOH 0.560 Slow Filter Crystalline [Pattern A] 54.6 2-MeTHF 1 60 Slow EvapCrystalline [Pattern D] 54 IPA with 0.5 60 Slow Filter Crystalline[Pattern A] 2% water

TABLE 13 Summary of binary solvent crystallization experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide (Startingmaterial, Pattern A used) Starting Material Rate of (Pattern PrimarySolvent Anti- Solvent Anti- A) Vol Temp Vol Solvent XRPD (mg) Solvent(mL) (° C.) Solvent (mL) Addition Isolation [Pattern] 84.5 EtOH 0.5 60Heptane 2.5 Slow Filter Crystalline [Pattern A] 53.7 Acetone 0.5 60Heptane 1.5 Slow Filter Crystalline [Pattern A] 82.2 EtOH 0.5 60 Heptane1.0 Fast Filter Crystalline [Pattern A] 56.1 Acetone 0.5 60 Heptane 1.0Fast Filter Crystalline [Pattern A]

Example 6. Scale-Up Experiments

Scale-up experiments were performed on a 300 mg scale by single solventfast cooling crystallizations in 2-MeTHF, EtOAc, and slurries in THF and0.5% Methyl Cellulose/2% Tween 80 in an attempt to isolate previouslyobserved Patterns D, E, B and C respectively for furthercharacterization. Experimental details and results are summarized inTables 14-15.

Fast cooling single solvent crystallization experiments were performedin 2-MeTHF and EtOAc (Table 14). A 48 position Chemglass reaction blockwas used for heating and stirring which were performed in 4 mL vials.Each vial was charged with approximately 300 mg of starting materialfitted with a magnetic stir bar. 6 mL of primary solvent was added andheated to 60° C. with stirring until dissolution achieved. Once fullydissolved the sample was (crashed cooled) transferred to an ice bath andseeded with a spatula tip full of Pattern D or E, followed by overnightequilibration at room temperature with stirring. Samples which affordedsolids after overnight equilibration were isolated by filtration andsamples that did not precipitate were evaporated under a gentle streamof nitrogen. All samples were dried overnight under vacuum at ambientconditions and XRPD analysis was performed to check for form change.

Slurry experiments were performed in THF and 0.5% Methyl Cellulose/2%Tween 80 (Table 15). A 48 position Chemglass reaction block was used forheating and stirring which were performed in 4 mL vials. Each vial wascharged with approximately 300 mg of starting material fitted with amagnetic stir bar. Slurry solvent was added up to 2 mL at ambientconditions and allowed to equilibrate for 30 minutes before adding aspatula tip full of Pattern B orC.

XRPD analysis of solids isolated from single solvent crystallizationsperformed in 2-MeTHF with fast and cooling profiles, afforded a uniqueXRPD pattern, designated as Pattern F (FIG. 4). Fast coolingcrystallization performed in EtOAc yielded crystalline solids consistentwith Pattern E by XRPD (FIG. 5). Slurry experiments performed in THF and0.5% Methyl Cellulose/2% Tween 80, were found to afford Patterns B and Crespectively following 24 hours of equilibration (FIG. 6-7).

TABLE 14 Summary of single solvent crystallization scale-up experimentsof (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.Starting material Seeding upon (Pattern A) Primary Solvent Temp Coolingcooling XRPD [mg] Solvent Vol (mL) (° C.) Rate (mg) Isolation [Pattern]312.0 2-MeTHF 6.0 60 Fast [~10] Evap Crystalline [Pattern F] 300.7 EtOAc6.0 60 Fast [~10] Filter Crystalline [Pattern E]

TABLE 15 Summary of slurry scale-up experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide Startingmaterial Primary (Pattern Solvent Slurry Slurry XRPD A) Vol Temp. For-Seeding upon [Pattern] (mg) Solvent (mL) (° C.) mation Slurry [mg] 24 hr305.7 THF 2 Ambient Yes [~10] Crystalline [Pattern B] 308.3 0.5% 2Ambient Yes [~1 0] Crystalline MC/2% [Pattern C] Tween 80 in water

Example 7. Competitive Slurries

Competitive slurry experiments of Patterns A, B, C, D, E and F wereinitiated in IPA, IPA/2% water and 0.5% Methyl Cellulose in water atambient conditions as summarized in Table 16. Approximately 100 mg ofthe starting material, Pattern A, was added to a glass vial fitted witha magnetic stir bar. Solvent was added to the vial and allowed to slurrywith Pattern A for 15 minutes before approximately 10-20 mg of eachrelative Pattern (B, C, D, E and F) was added to each vial. The sampleswere allowed to equilibrate with stirring and following 24 hours orequilibration, XRPD analysis showed solids isolated from slurry in 0.5%Methyl Cellulose in water was a mixture of Patterns A/C (FIG. 8). Allother solids isolated from IPA and IPA/2% water were consistent with thestarting material, Pattern A (FIG. 9). However, following 7 days ofequilibration full conversion to Pattern C was observed from slurry in0.5% Methyl Cellulose in water (FIG. 8), where as solids isolated fromcompetitive slurry in IPA and IPA/2% water were consistent with PatternA (FIG. 9).

TABLE 16 A summary of competitive slurry experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide in 2 mL of solvent.Pattern (mg) Pattern Pattern Pattern Pattern Pattern Pattern XRPDSolvent A B C D E F 24 hrs 7 days IPA 101.9* ~10 ~10 ~10 ~10 ~10 A AIPA:water 107.3* ~10 ~10 ~10 ~10 ~10 A A (98:2) 0.5% 103.9* ~10 ~10 ~10~10 ~10 A/C C MC/water *Used to saturate the solvent and make a thinslurry of Pattern A

Example 8. Elevated Humidity Studies

7 day elevated aqueous humidity experiments were performed on allPatterns A, B, C, D, E and F at ambient conditions at >95% RH (Table17). Approximately 30 mg of each pattern was weighed into a 4 ml glassvial. The uncovered 4 ml vial was inserted into a 20 ml scintillationvial half filled with water and capped. Following 24 hours ofequilibration, visual inspection was performed to check for changes inphysical appearance, however no change was observed. Following 7 days ofequilibration at elevated humidity, the sample showed no physicalchanges and was analyzed by XRPD to check for form. Patterns A, C and Eshowed no change in form after 7 days of equilibration; however PatternB was converted to a mixture of Patterns A/B. Pattern D was found toconvert to a mixture of Patterns D/B and Pattern F was converted toPattern E at >95% RH. All experimental details and results aresummarized in Table 17.

TABLE 17 Summary of 7 day elevated aqueous humidity experiments of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide XRPD Storage Visual Observations[Pattern] [mg] Conditions Initial Pattern 1 day 7 day 7 day ~30 >95% RHat A Yellow Yellow Crystalline Ambient solids Solids [Pattern A]~30 >95% RH at B Yellow Yellow Crystalline Ambient solids solids[Mixture of Patterns A/B] ~30 >95% RH at C Yellow Yellow CrystallineAmbient solids solids [Pattern C] ~30 >95% RH at D Yellow YellowCrystalline Ambient solids solids [Mixture of Patterns D/B] ~30 >95% RHat E Yellow Yellow Crystalline Ambient solids solids [Pattern E]~30 >95% RH at F Yellow Yellow Crystalline Ambient solids solids[Pattern E]

Example 9. Grinding Experiments

Grinding experiments of the starting material were performed by drygrinding and solvent drop grinding in IPA and water utilizing a mortarand pestle (Table 18). Following light grinding pattern A, no change inthe crystal form was observed by XRPD analysis.

TABLE 18 Summary of Grinding Experiments of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide Sample Wt.(mg) Solvent Grinding Conditions XRPD 30-50 — yes RT Pattern A 30-50 IPA(2 drops) yes RT Pattern A 30-50 H2O (2 drops) yes RT Pattern A

Example 10. Aqueous Solubility

Aqueous solubility of Patterns A, B, C, E and F was performed using anAgilent HPLC system. Approximately 10-20 mg of each form was charged toa 2 mL glass vial loaded with a magnetic stir bar and added 2 mL ofwater. The samples were allowed to stir overnight at ambient conditions.Following 24 hours of equilibration the samples were centrifuged anddecanted into HPLC vials. A calibration curve was generated based onPattern A in MeOH at 0.05, 0.1, 0.5 and 1.0 mg/mL. Following injectionof the standard curve, the samples were run as is. All experimentaldetails and results are summarized in Table 19.

TABLE 19 Summary of Aqueous Solubility experiments of (R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideWater Pat- Solubility (mg) (mL) tern Designated Form % AUC (mg/mL) 15.02 F 2-MeTHF Solvate Form VI 6813 1.36 12.1 2 E Anhydrate Form II 66021.32 12.3 2 B THF Solvate Form IV 6514 1.30 13.5 2 C Hydrate Form III6646 1.32 21.5 2 A Anhydrate Form 1 8729 1.74

Example 11. Characterization of Forms

Solid form characterization of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,polymorphic forms was completed by XRPD, DSC, TGA, ¹H NMR, Karl Fischer,optical microscopy, and moisture sorption. Results are summarized inTable 1.

Pattern A (Anhydrate, Form I)

Utilizing the starting material as noted in Example 2, XRPD analysis ofthe yellow colored starting material was found to afford a crystallinepattern, designated as Pattern A (FIG. 10). The crystallinity observedby XRPD was confirmed by the exhibition of birefringence observed byoptical microscopy. The morphology of the crystals was determined to beirregularly shaped with some aggregation as shown in FIG. 11.

Thermal analysis by DSC showed a single endothermic event at peak of152.9° C., followed by degradation after 200° C. (FIG. 12).

TGA analysis showed no weight loss between 45-160° C., however weightloss due to decomposition was observed from 160-300° C. (FIG. 13).Minimal moisture content was confirmed by Karl Fischer analysis whichshowed the materials to contain approximately 0.12 wt % water.

Further analysis by ¹H NMR showed the starting material to be consistentwith structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand to contain 0.28 wt % residual IPA. See FIG. 40.

Moisture sorption analysis of the starting material was performed byequilibrating the sample at 25° C. and 50% RH to simulate ambient labconditions. Humidity then decreased to 0% RH, increased from 0 to 95%RH, reduced from 95 to 0% RH, increased from 0 to 95% RH and thendecreased from 95 to 50% RH. Each point represents the estimatedasymptotic weight for each humidity or weight. The starting material wasfound to non-hygroscopic, adsorbing 0.1% water at 90% RH. No hysteresiswas observed upon desorption (FIG. 14). XRPD analysis of samplefollowing moisture sorption analysis was found to be consistent with thestarting material, Pattern A.

Pattern E (Anhydrate, Form II)

Pattern E (Anhydrate, Form II) was observed during single solventcrystallizations (Examples 5 and 6) using a fast cooling profile at the50 mg scale and again at the 300 mg scale-up. XRPD analysis of thesolids were found to afford a unique crystalline pattern, designated asPattern E (FIG. 15). The crystallinity observed by XRPD was confirmed bythe exhibition of birefringence observed by optical microscopy. Themorphology of the crystals was determined to be irregularly shaped withsome aggregation as shown in FIG. 16.

Thermal analysis of the 50 mg scale lot by DSC showed a two endothermicevents at peak of 133.9° C. and 151.3° C., followed by degradation after200° C. (FIG. 17).

TGA analysis of the 50 mg scale lot, showed a 0.4% weight loss between120-140° C., likely attributed to the loss of EtOAc, followed bydecomposition (FIG. 18). Minimal moisture content was confirmed by KarlFischer analysis which showed the materials to contain approximately 0.1wt % water.

Further analysis of Pattern E (from the 300 mg scale lot) by ¹H NMRshowed the material to be consistent with structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand to contain 0.4 wt % residual EtOAc. See FIG. 44.

Moisture sorption analysis of Pattern E (from the 300 mg scale lot) wasperformed by equilibrating the sample at 25° C. and 50% RH to simulateambient lab conditions. Humidity was then decreased to 0% RH, increasedfrom 0 to 95% RH, reduced from 95 to 0% RH, increased from 0 to 95% RHand then decreased from 95 to 50% RH. Each point represents theestimated asymptotic weight for each humidity or weight. Pattern E wasfound to non-hygroscopic, adsorbing 0.2% water at 95% RH. No hysteresiswas observed upon desorption (FIG. 19). XRPD analysis of samplefollowing moisture sorption analysis was found to be consistent withPattern E.

Pattern C (Hydrate, Form III)

Pattern C (Hydrate, Form III) was observed during short term slurry in0.5% Methyl Cellulose/2% Tween 80 at the 50 mg scale and again at the300 mg scale-up (Examples 3 and 6, respectively). XRPD analysis of thesolids were found to afford a unique crystalline pattern, designated asPattern C (FIG. 20). The crystallinity observed by XRPD was confirmed bythe exhibition of birefringence observed by optical microscopy. Themorphology of the crystals was determined to be irregularly shaped withsome aggregation as shown in FIG. 21.

Thermal analysis of the 50 mg lot by DSC showed two endothermic eventsat peaks of 72° C. and 150.7° C., followed by degradation after 200° C.(FIG. 22).

TGA analysis of the 50 mg lot showed a 2.5% weight loss between 20-60°C., followed by a 2.3% wt. loss from 60-125° C., likely attributed todehydration, followed by decomposition (FIG. 23). Moisture content wasconfirmed by Karl Fischer analysis which showed the materials to containapproximately 4.3 wt % water, slightly lower than a monohydrate.

Further analysis of Pattern C (300 mg lot) by ¹H NMR showed the materialto be consistent with structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.See FIG. 42.

Moisture sorption analysis of Pattern C (300 mg lot) was performed byequilibrating the sample at 25° C. and 50% RH to simulate ambient labconditions. Humidity was then decreased to 0% RH, increased from 0 to95% RH, reduced from 95 to 0% RH, increased from 0 to 95% RH and thendecreased from 95 to 50% RH. Each point represents the estimatedasymptotic weight for each humidity or weight. Pattern C was found to beslightly hygroscopic, adsorbing 2% water at 95% RH. This increased thetotal water to about 6% which is the water content of mono-hydrate.However, upon reducing the relative humidity, the solid lost its water.Therefore this could be a channel hydrate. No hysteresis was observedupon desorption (FIG. 24). XRPD analysis of sample following moisturesorption analysis was found to be consistent with Pattern C.

Pattern B (THF Solvate, Form IV)

Pattern B (THF Solvate, Form IV) was observed during short term slurryin THF at the 50 mg scale and again at the 300 mg scale-up (Examples 3and 6 respectively). XRPD analysis of the solids were found to afford aunique crystalline pattern, designated as Pattern B (FIG. 25). Thecrystallinity observed by XRPD was confirmed by the exhibition ofbirefringence observed by optical microscopy. The morphology of thecrystals was determined to be needle shaped with some aggregation asshown in FIG. 26.

Thermal analysis of the 50 mg lot, by DSC showed three endothermicevents at peak of 70.5, 89.1° C. and 149.7° C., followed by degradationafter 200° C. (FIG. 27).

TGA analysis of the 300 mg lot, showed a 4.7% weight loss between25-115° C., likely attributed to the loss of THF, followed bydecomposition (FIG. 28). Moisture content was confirmed by Karl Fischeranalysis which showed the materials to contain approximately 0.3 wt %water.

Further analysis of Pattern B (300 mg lot) by ¹H NMR showed the materialto be consistent with structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand contain 6.9 wt % residual THF. See FIG. 41.

Moisture sorption analysis of Pattern B (300 mg lot) was performed byequilibrating the sample at 25° C. and 50% RH to simulate ambient labconditions. Humidity then decreased to 0% RH, increased from 0 to 95%RH, reduced from 95 to 0% RH, increased from 0 to 95% RH and thendecreased from 95 to 50% RH. Each point represents the estimatedasymptotic weight for each humidity or weight. Pattern B was found to benon-hygroscopic, showing weight loss likely due to release of residualTHF (FIG. 29). The major weight loss at the beginning was due to loss ofsolvent. XRPD analysis of sample following moisture sorption analysiswas found to be consistent with Pattern A.

Pattern D (2-MeTHF Solvate, Form V)

Pattern D (2-MeTHF Solvate, Form V) was observed during evaporativecrystallizations in 2-MeTHF (Examples 4 and 5). XRPD analysis of thesolids were found to afford a unique crystalline pattern, designated asPattern D (FIG. 30).

Thermal analysis of the Pattern D from Example 5 (slow cooling), by DSCshowed four endothermic events at peaks of 67-2, 92.2, 132.6 and 150.6°C., followed by degradation after 220° C. (FIG. 31).

TGA analysis of Example 5 slow cooling lot, showed a 2.7% weight lossbetween 40-60° C. followed by a 5.3% weight loss from 60-115° C., likelyattributed to the loss of 2-MeTHF, followed by decomposition (FIG. 32).

Further analysis of Pattern D (Example 5 slow cooling lot) by ¹H NMRshowed the material to be consistent with structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand contain 6.1 wt % residual 2-MeTHF. See FIG. 43.

Pattern F (2-MeTHF Solvate, Form VI)

Pattern F (2-MeTHF Solvate, Form VI) was observed during single solventcrystallization scale-up experiments 2-MeTHF at the 300 mg scale(Example 6). XRPD analysis of the solids were found to afford a uniquecrystalline pattern, designated as Pattern F (FIG. 33). Thecrystallinity observed by XRPD was confirmed by the exhibition ofbirefringence observed by optical microscopy. The morphology of thecrystals was determined to be plate shaped with some aggregation asshown in FIG. 34.

Thermal analysis of Pattern F (from Example 6) by DSC showed threeendothermic events at peaks of 93.2, 135.2° C. and 151.0° C., followedby degradation after 220° C. (FIG. 35).

TGA analysis of Pattern F (from Example 6), showed a 1.1% weight lossbetween 30-110° C., followed by a 0.2% weight loss from 110-160° C.,likely attributed to the loss of 2-MeTHF, followed by decomposition(FIG. 36). Moisture content was confirmed by Karl Fischer analysis whichshowed the materials to contain approximately 0.1 wt % water.

Further analysis of Pattern F (from Example 6) by ¹H NMR showed thematerial to be consistent with structure of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamideand contain 3.9 wt % residual 2-MeTHF. See FIG. 45.

Moisture sorption analysis of Pattern F (from Example 6) was performedby equilibrating the sample at 25° C. and 50% RH to simulate ambient labconditions. Humidity was then decreased to 0% RH, increased from 0 to95% RH, reduced from 95 to 0% RH, increased from 0 to 95% RH and thendecreased from 95 to 50% RH. Each point represents the estimatedasymptotic weight for each humidity or weight. Pattern F was found to benon-hygroscopic, showing weight loss likely due to release of residual2-MeTHF (FIG. 37). The major weight loss at the beginning is due to lossof solvent. XRPD analysis of the sample following moisture sorptionanalysis was found to be consistent with Pattern E (FIG. 15).

Example 12. Screening Compounds of the Invention in Human DermalFibroblasts from Friedreich's Ataxia Patients

An initial screen was performed to identify compounds effective for theamelioration of redox disorders. Test samples were tested for theirability to rescue FRDA fibroblasts stressed by addition ofL-buthionine-(S,R)-sulfoximine (BSO), as described in Jauslin et al.,Hum. Mol. Genet. 11(24):3055 (2002), Jauslin et al., FASEB J. 17:1972-4(2003), and International Patent Application WO 2004/003565. Humandermal fibroblasts from Friedreich's Ataxia patients have been shown tobe hypersensitive to inhibition of the de novo synthesis of glutathione(GSH) with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor ofGSH synthetase (Jauslin et al., Hum. Mol. Genet. 11(24):3055 (2002)).

MEM (a medium enriched in amino acids and vitamins, catalog no.1-31F24-I) and Medium 199 (M199, catalog no. 1-21F22-I) with Earle'sBalanced Salts, without phenol red, were purchased from Bioconcept.Fetal Calf Serum was obtained from PAA Laboratories. Basic fibroblastgrowth factor and epidermal growth factor were purchased from PeproTech.Penicillin-streptomycin-glutamine mix, L-buthionine (S,R)-sulfoximine,and insulin from bovine pancreas were purchased from Sigma. Calcein AMwas purchased from Anaspec. Cell culture medium was made by combining125 ml M199 EBS, 50 ml Fetal Calf Serum, 100 U/ml penicillin, 100microgram/ml streptomycin, 2 mM glutamine, 10 microgram/ml insulin, 10ng/ml EGF, and 10 ng/ml bFGF; MEM EBS was added to make the volume up to500 ml. During the course of the experiments, this solution was storedat +4° C. The cells were obtained from the Coriell Cell Repositories(Camden, N.J.; repository number GM04078) and grown in 10 cm tissueculture plates. Every third day, they were split at a 1:3 ratio.

The test samples were supplied in 1.5 ml glass vials. The compounds werediluted with DMSO, ethanol or PBS to result in a 5 mM stock solution.Once dissolved, they were stored at −20° C.

Test samples were screened according to the following protocol:

A culture with FRDA fibroblasts was started from a 1 ml vial withapproximately 500,000 cells stored in liquid nitrogen. Cells werepropagated in 10 cm cell culture dishes by splitting every third day ina ratio of 1:3 until nine plates were available. Once confluent,fibroblasts were harvested. For 54 micro titer plates (96 well-MTP) atotal of 14.3 million cells (passage eight) were re-suspended in 480 mlmedium, corresponding to 100 microliters medium with 3,000 cells/well.The remaining cells were distributed in 10 cm cell culture plates(500,000 cells/plate) for propagation. The plates were incubatedovernight at 37° C. in a atmosphere with 95% humidity and 5% CO2 toallow attachment of the cells to the culture plate.

10% DMSO (242.5 microliters) was added to a well of the microtiterplate. The test compounds were unfrozen, and 7.5 microliters of a 5 mMstock solution was dissolved in the well containing 242.5 microliters of10% DMSO, resulting in a 150 micromolar master solution. Serialdilutions from the master solution were made. The period between thesingle dilution steps was kept as short as possible (generally less than30 seconds). At least 4 hours after attachement into MTP, cells werethen treated with the various compound dilutions.

Plates were kept overnight in the cell culture incubator. The next day,a solution containing BSO was added to the wells, in a manner similar tothat described in Jauslin et al., Hum. Mol. Genet. 11(24):3055 (2002),Jauslin et al., FASEB J. 17:1972-4 (2003), and International PatentApplication WO 2004/003565. Forty-eight hours later, three plates wereexamined under a phase-contrast microscope to verify that the cells inthe negative control (wells E1-H1) were clearly dead. The medium fromall plates was discarded, and the remaining liquid was removed by gentlytapping the plate inversed onto a paper towel. The plates were washedtwice with 100 uL of PBS containing Calcium and Magnesium.

100 microliters of PBS+Ca+Mg containing 1.2 micromolar Calcein AM werethen added to each well. The plates were incubated for 30 minutes at 37C. After that time fluorescence (excitation/emission wavelengths of 485nm and 525 nm, respectively) was read on a Gemini fluorescence reader.Data was imported into Microsoft Excel (EXCEL is a registered trademarkof Microsoft Corporation for a spreadsheet program) and ExcelFit wasused to calculate the EC50 concentration for each compound.

The compounds were tested three times, i.e., the experiment wasperformed three times, the passage number of the cells increasing by onewith every repetition.

The solvents (DMSO, ethanol, PBS) neither had a detrimental effect onthe viability of non-BSO treated cells nor did they have a beneficialinfluence on BSO-treated fibroblasts even at the highest concentrationtested (1%). None of the compounds showed auto-fluorescence. Theviability of non-BSO treated fibroblasts was set as 100%, and theviability of the BSO- and compound-treated cells was calculated asrelative to this value.

The following table summarizes the EC50 for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

Disorder EC50 Friedrich's Ataxia +++ +++ indicates less than 100 nM

Example 13. Screening(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein Fibroblasts from Patients Having Various Oxidative Stress Disorders

(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidewas tested using a screen similar to the one described in Example 12,and substituting FRDA cells with cells from patients having otheroxidative stress disorders.

The following table summarizes the EC50 for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidefor various disorders.

Disorder Cell Line Tested EC50 Leigh Syndrome Coriell Cell Repositories+++ (Camden, NJ; repository number GMOI503A) Leber's Hereditary OpticCoriell Cell Repositories +++ Neuropathy (LHON) (Camden, NJ; repositorynumber GM03858) Parkinson's Disease Coriell Cell Repositories ++(Camden, NJ; repository number AG20439) Huntington's Disease CoriellCell Repositories +++ (Camden, NJ; repository number GM 04281) Rett'sDisorder Coriell Cell Repositories +++ (Camden, NJ; repository numberGM-17567) CoQ10 Deficiency From patients having a +++ CoQ2 mutationAmyotrophic Lateral Sclerosis Coriell Cell Repositories +++ (ALS)(Camden, NJ; repository number ND29523) +++ indicates less than 100 nM,++ indicates 100-500 nM

Example 14. Screening(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidefor Protection from Cisplatin-Induced Ototoxicity

The conditionally immortalized auditory HEI-OC1 cells from long-termcultures of transgenic mice Immortomouse™ cochleas as described inKalinec, G. et al., Audiol. Nerootol. 2003; 8, 177-189/. were maintainedin high glucose Dulbecco's modified Eagle medium (DMEM) containing 10%FBS under permissive conditions, 33° C., 10% CO2. Cells were pretreatedovernight with(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,and apoptosis was detected by caspase3/7 activity after 24 hours of 50uM cisplatin incubation. Cells incubated in diluent alone were thecontrols.

The following table summarizes the EC25 for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.

Disorder EC25 Cisplatin-induced ototoxicity +++ of auditory cells +++indicates less than 100 micromolar

Example 15. Screening Polymorphic and Amorphous Compositions of theInvention in Fibroblasts from Patients

Polymorphic and amorphous compositions of the invention are tested usingscreens similar to the ones described in Examples 12-14, substitutingthe polymorphic or amorphous form for(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide.Polymorphic and amorphous compositions of the invention are also testedusing screens similar to the ones described in Examples 12-14, and whereappropriate substituting the FRDA cells or other cell lines with cellsobtained from patients having an oxidative stress disorder describedherein (e.g. MERRF, MELAS, KSS, Alzheimer's disease, a pervasivedevelopment disorder (such as autism), etc). The compositions are testedfor their ability to rescue human dermal fibroblasts from these patientsfrom oxidative stress or for their ability to protect cells fromcisplatin-induced toxicity.

Example 16. Administration of Compositions of the Invention

A composition of the invention is presented in a capsule containing 300mg of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamidein a pharmaceutically acceptable carrier. A capsule is taken orally,once a day.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein by an identifyingcitation are hereby incorporated herein by reference in their entirety.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

What is claimed is:
 1. A polymorph of an anhydrateor a hydrate of(R)-2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanamide,wherein the polymorph is selected from Form III (hydrate), or Form II(anhydrate); wherein a powder X-ray diffraction pattern for polymorphForm III comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by±0.2: 14.02,15.23, and 21.10; and wherein a powder X-ray diffraction pattern forpolymorph Form II comprises characteristic peaks at least at thefollowing angular positions, wherein the angular positions may vary by±0.2: 9.63, 11.33, and 19.33.
 2. The polymorph of claim 1, wherein thepolymorph is Form III, wherein a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2:14.02, 15.23, and 21.10.
 3. The polymorph of claim 1, wherein thepolymorph is Form III, wherein a powder X-ray diffraction pattern forthe polymorph comprises characteristic peaks at least at the followingangular positions, wherein the angular positions may vary by ±0.2: 9.16,14.02, 15.23, 21.10, and 22.69.
 4. The polymorph of claim 1, wherein thepolymorph is Form III, wherein the polymorph has a powder x-raydiffraction pattern substantially as shown in a) orb) of FIG.
 20. 5. Acomposition comprising the polymorph of claim 2, wherein at least about95% by mole of the composition is the polymorph Form III, exclusive ofany solvents, carriers or excipients.
 6. The polymorph of claim 1,wherein the polymorph is Form III, having a differential scanningcalorimetry (DSC) thermogram substantially as shown in FIG.
 22. 7. Thepolymorph of claim 6, wherein the DSC thermogram comprises twoendothermic peaks at about 72.0 and 150.7° C.
 8. The polymorph of claim1, wherein the polymorph is Form III, having a thermogravimetricanalysis (TGA) thermogram substantially as shown in FIG.
 23. 9. Thepolymorph of claim 1, wherein the polymorph is Form III, having a ¹H NMRspectrum substantially as shown in FIG.
 42. 10. A pharmaceuticalcomposition comprising the polymorph of claim 2, and a pharmaceuticallyacceptable carrier.
 11. The polymorph of claim 1, wherein the polymorphis Form II, wherein a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by ±0.2: 9.63, 11.33,and 19.33.
 12. The polymorph of claim 1, wherein the polymorph is FormII, wherein a powder X-ray diffraction pattern for the polymorphcomprises characteristic peaks at least at the following angularpositions, wherein the angular positions may vary by ±0.2: 9.63, 10.85,11.33, 13.47, and 19.33.
 13. The polymorph of claim 1, wherein thepolymorph is Form II, wherein the polymorph has a powder x-raydiffraction pattern substantially as shown in a) of FIG.
 15. 14. Acomposition comprising the polymorph of claim 11, wherein at least about95% by mole of the composition is the polymorph Form II, exclusive ofany solvents, carriers or excipients.
 15. The polymorph of claim 1,wherein the polymorph is Form II, having a differential scanningcalorimetry (DSC) thermogram substantially as shown in FIG.
 17. 16. Thepolymorph of claim 15, wherein the DSC thermogram comprises twoendothermic peaks at about 133.9 and 151.3° C.
 17. The polymorph ofclaim 1, wherein the polymorph is Form II, having a thermogravimetricanalysis (TGA) thermogram substantially as shown in FIG.
 18. 18. Thepolymorph of claim 1, wherein the polymorph is Form II, having a ¹H NMRspectrum substantially as shown in FIG.
 44. 19. A pharmaceuticalcomposition comprising the polymorph of claim 11, and a pharmaceuticallyacceptable carrier.
 20. A method of treating or suppressing an oxidativestress disorder, comprising administering to an individual in needthereof a therapeutically effective amount of the polymorph of claim 1,wherein the oxidative stress disorder is selected from the groupconsisting of: a mitochondrial disorder; an inherited mitochondrialdisease; Alpers Disease; Barth syndrome; a Beta-oxidation Defect;Carnitine-Acyl-Carnitine Deficiency; Carnitine Deficiency; a CreatineDeficiency Syndrome; Co-Enzyme Q10 Deficiency; Complex I Deficiency;Complex II Deficiency; Complex III Deficiency; Complex IV Deficiency;Complex V Deficiency; COX Deficiency; chronic progressive externalophthalmoplegia (CPEO); CPT I Deficiency; CPT II Deficiency;Friedreich's Ataxia (FA); Glutaric Aciduria Type II; Kearns-SayreSyndrome (KSS); Lactic Acidosis; Long-Chain Acyl-CoA DehydrogenaseDeficiency (LCAD); LCHAD; Leigh Syndrome; Leigh-like Syndrome; Leber'sHereditary Optic Neuropathy (LHON); Lethal Infantile Cardiomyopathy(LIC); Luft Disease; Multiple Acyl-CoA Dehydrogenase Deficiency (MAD);Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCAD); MitochondrialMyopathy, Encephalopathy, Lactacidosis, Stroke (MELAS); MyoclonicEpilepsy with Ragged Red Fibers (MERRF); Mitochondrial Recessive AtaxiaSyndrome (MIRAS); Mitochondrial Cytopathy, Mitochondrial DNA Depletion;Mitochondrial Encephalopathy; Mitochondrial Myopathy;Myoneurogastrointestinal Disorder and Encephalopathy (MNGIE);Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP); Pearson Syndrome;Pyruvate Carboxylase Deficiency; Pyruvate Dehydrogenase Deficiency; aPOLG Mutation; a Respiratory Chain Disorder; Short-Chain Acyl-CoADehydrogenase Deficiency (SCAD); SCHAD; Very Long-Chain Acyl-CoADehydrogenase Deficiency (VLCAD); a myopathy; cardiomyopathy;encephalomyopathy; a neurodegenerative disease; Parkinson's disease;Alzheimer's disease; amyotrophic lateral sclerosis (ALS); a motor neurondisease; a neurological disease; epilepsy; an age-associated disease;macular degeneration; diabetes; metabolic syndrome; cancer; braincancer; a genetic disease; Huntington's Disease; a mood disorder;schizophrenia; bipolar disorder; a pervasive developmental disorder;autistic disorder; Asperger's syndrome; childhood disintegrativedisorder (CDD); Rett's disorder; PDD-not otherwise specified (PDD-NOS);a cerebrovascular accident; stroke; a vision impairment; opticneuropathy; dominant inherited juvenile optic atrophy; optic neuropathycaused by a toxic agent; glaucoma; Stargardt's macular dystrophy;diabetic retinopathy; diabetic maculopathy; retinopathy of prematurity;ischemic reperfusion-related retinal injury; oxygen poisoning; ahaemoglobinopathy; thalassemia; sickle cell anemia; seizures; ischemia;renal tubular acidosis; attention deficit/hyperactivity disorder (ADHD);a neurodegenerative disorder resulting in hearing or balance impairment;Dominant Optic Atrophy (DOA); Maternally inherited diabetes and deafness(MIDD); chronic fatigue; contrast-induced kidney damage;contrast-induced retinopathy damage; Abetalipoproteinemia; retinitispigmentosum; Wolfram's disease; Tourette syndrome; cobalamin c defect;methylmalonic aciduria; glioblastoma; Down's syndrome; acute tubularnecrosis; a muscular dystrophy; a leukodystrophy; ProgressiveSupranuclear Palsy; spinal muscular atrophy; hearing loss; noise inducedhearing loss; traumatic brain injury; Juvenile Huntington's Disease;Multiple Sclerosis; NGLY1; Multiple System Atrophy;Adrenoleukodystrophy; and Adrenomyeloneuropathy.